Plant regulatory elements and uses thereof

ABSTRACT

The invention provides recombinant DNA molecules and constructs, as well as their nucleotide sequences, useful for modulating gene expression in plants. The invention also provides transgenic plants, plant cells, plant parts, and seeds comprising the recombinant DNA molecules operably linked to heterologous transcribable DNA molecules, as are methods of their use.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application No.62/714,228, filed Aug. 3, 2018, which is herein incorporated byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“38-21-62691-0001_Seqlist_ST25.txt”, is 31,060 bytes (as measured inMicrosoft Windows®), was created on Jul. 2, 2019, and is filed herewithby electronic submission and incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to the field of plant molecular biology and plantgenetic engineering. More specifically, the invention relates to DNAmolecules useful for modulating gene expression in plants.

BACKGROUND

Regulatory elements are genetic elements that regulate gene activity bymodulating the transcription of an operably linked transcribable DNAmolecule. Such elements may include promoters, leaders, introns, and 3′untranslated regions and are useful in the field of plant molecularbiology and plant genetic engineering.

SUMMARY OF THE INVENTION

The invention provides novel synthetic gene regulatory elements for usein plants. The invention also provides recombinant DNA moleculesconstructs comprising the regulatory elements. The present inventionalso provides transgenic plant cells, plants, and seeds comprising theregulatory elements. In one embodiment, the regulatory elements areoperably linked to a transcribable DNA molecule. In certain embodiments,the transcribable DNA molecule may be heterologous with respect to theregulatory sequence. Thus, a regulatory element sequence provided by theinvention may, in particular embodiments, be defined as operably linkedto a heterologous transcribable DNA molecule. The present invention alsoprovides methods of using the regulatory elements and making and usingthe recombinant DNA molecules comprising the regulatory elements, andthe transgenic plant cells, plants, and seeds comprising the regulatoryelements operably linked to a transcribable DNA molecule.

Thus, in one aspect, the invention provides a recombinant DNA moleculecomprising a DNA sequence selected from the group consisting of: (a) asequence with at least about 85 percent sequence identity to any of SEQID NOs:1-19 and SEQ ID NO:26; (b) a sequence comprising any of SEQ IDNOs:1-19 and SEQ ID NO:26; and (c) a fragment of any of SEQ ID NOs:1-19and SEQ ID NO:26, wherein the fragment has gene-regulatory activity;wherein the sequence is operably linked to a heterologous transcribableDNA molecule. By “heterologous transcribable DNA molecule,” it is meantthat the transcribable DNA molecule is heterologous with respect to thepolynucleotide sequence to which it is operably linked. In specificembodiments, the recombinant DNA molecule comprises a DNA sequencehaving at least about 85 percent, at least about 86 percent, at leastabout 87 percent, at least about 88 percent, at least about 89 percent,at least about 90 percent, at least 91 percent, at least 92 percent, atleast 93 percent, at least 94 percent, at least 95 percent, at least 96percent, at least 97 percent, at least 98 percent, or at least 99percent sequence identity to the DNA sequence of any of SEQ ID NOs:1-19and SEQ ID NO:26. In particular embodiments, the DNA sequence comprisesa regulatory element. In some embodiments, the regulatory elementcomprises a promoter. In still other embodiments, the regulatory elementcomprises an intron. In still other embodiments, the regulatory elementcomprises a 3′ UTR. In still other embodiments, the heterologoustranscribable DNA molecule comprises a gene of agronomic interest, suchas a gene capable of providing herbicide resistance in plants, or a genecapable of providing plant pest resistance in plants. In still otherembodiments, the heterologous transcribable DNA molecule comprises asequence encoding a small RNA, such as a dsRNA, an miRNA, or siRNA. Instill other embodiments, the invention provides a construct comprising arecombinant DNA molecule as provided herein.

In another aspect, provided herein are transgenic plant cells comprisinga recombinant DNA molecule comprising a DNA sequence selected from thegroup consisting of: (a) a sequence with at least about 85 percentsequence identity to any of SEQ ID NOs:1-19 and SEQ ID NO:26; (b) asequence comprising any of SEQ ID NOs:1-19 and SEQ ID NO:26; and (c) afragment of any of SEQ ID NOs:1-19 and SEQ ID NO:26, wherein thefragment has gene-regulatory activity; wherein the DNA sequence isoperably linked to a heterologous transcribable DNA molecule. In certainembodiments, the transgenic plant cell is a monocotyledonous plant cell.In other embodiments, the transgenic plant cell is a dicotyledonousplant cell.

In still yet another aspect, further provided herein is a transgenicplant, or part thereof, comprising a recombinant DNA molecule comprisinga DNA sequence selected from the group consisting of: a) a sequence withat least 85 percent sequence identity to any of SEQ ID NOs:1-19 and SEQID NO:26; b) a sequence comprising any of SEQ ID NOs:1-19 and SEQ IDNO:26; and c) a fragment of any of SEQ ID NOs:1-19 and SEQ ID NO:26,wherein the fragment has gene-regulatory activity; wherein the sequenceis operably linked to a heterologous transcribable DNA molecule. Inspecific embodiments, the transgenic plant is a progeny plant of anygeneration that comprises the recombinant DNA molecule. A transgenicseed comprising the recombinant DNA molecule that produces such atransgenic plant when grown is also provided herein.

In another aspect, the invention provides a method of producing acommodity product comprising obtaining a transgenic plant or partthereof containing a recombinant DNA molecule of the invention andproducing the commodity product therefrom. In one embodiment, thecommodity product is seeds, processed seeds, protein concentrate,protein isolate, starch, grains, plant parts, seed oil, biomass, flourand meal.

In still yet another aspect, the invention provides a method ofproducing a transgenic plant comprising a recombinant DNA molecule ofthe invention comprising transforming a plant cell with the recombinantDNA molecule of the invention to produce a transformed plant cell andregenerating a transgenic plant from the transformed plant cell.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a DNA sequence of a synthetic regulatory expressionelement group (EXP), EXP-Zm.GSP850 comprising a synthetic promoter(P-Zm.GSP850.nno:4), operably linked 5′ to a synthetic leader(L-Zm.GSP850.nno:3).

SEQ ID NO:2 is a DNA sequence of a synthetic promoter,P-Zm.GSP850.nno:4.

SEQ ID NO:3 is a DNA sequence of a synthetic leader, L-Zm.GSP850.nno:3.

SEQ ID NO:4 is a DNA sequence of a synthetic EXP,EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 comprising a synthetic promoter(P-Zm.GSP850.nno:4), operably linked 5′ to a synthetic leader(L-Zm.GSP850.nno:3), operably linked 5′ to a synthetic intron(I-Zm.GSI153.nno: 1).

SEQ ID NO:5 is a DNA sequence of a synthetic intron, I-Zm.GSI153.nno:1.

SEQ ID NO:6 is a DNA sequence of a synthetic EXP, EXP-Zm.GSP990comprising a synthetic promoter (P-Zm.GSP990.nno:2), operably linked 5′to a synthetic leader (L-Zm.GSP990.nno: 1).

SEQ ID NO:7 is a DNA sequence of a synthetic promoter,P-Zm.GSP990.nno:2.

SEQ ID NO:8 is a DNA sequence of a synthetic leader, L-Zm.GSP990.nno:1.

SEQ ID NO:9 is a DNA sequence of a synthetic EXP,EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 comprising a synthetic promoter(P-Zm.GSP990.nno:2), operably linked 5′ to a synthetic leader(L-Zm.GSP990.nno:1), operably linked 5′ to a synthetic intron(I-Zm.GSI197.nno:1).

SEQ ID NO:10 is a DNA sequence of a synthetic intron, I-Zm.GSI197.nno:1.

SEQ ID NO:11 is a DNA sequence of a synthetic EXP,EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 comprising a synthetic promoter(P-Zm.GSP850.nno:4), operably linked 5′ to a synthetic leader(L-Zm.GSP850.nno:3), operably linked 5′ to a synthetic intron(I-Zm.GSI140.nno:1).

SEQ ID NO:12 is a DNA sequence of a synthetic intron, I-Zm.GSI140.nno:1.

SEQ ID NO:13 is a DNA sequence of a synthetic 3′ UTR, T-Zm.GST9.nno:2.

SEQ ID NO:14 is a DNA sequence of a synthetic 3′ UTR, T-Zm.GST18.nno:2.

SEQ ID NO:15 is a DNA sequence of a synthetic EXP,EXP-Zm.GSP850.nno+Zm.DnaK:1 comprising a synthetic promoter(P-Zm.GSP850.nno:4), operably linked 5′ to a synthetic leader(L-Zm.GSP850.nno:3), operably linked 5′ to an intron (I-Zm.DnaK:1).

SEQ ID NO:16 is DNA sequence of a synthetic EXP,EXP-Zm.GSP990.nno+Zm.DnaK:1 comprising a synthetic promoter(P-Zm.GSP990.nno:2), operably linked 5′ to a synthetic leader(L-Zm.GSP990.nno:1), operably linked 5′ to an intron (I-Zm.DnaK: 1).

SEQ ID NO:17 is a DNA sequence of a synthetic enhancer, E-Zm.GSP850which is derived from the synthetic promoter, P-Zm.GSP850.nno:4.

SEQ ID NO:18 is a DNA sequence of a synthetic enhancer, E-Zm.GSP990which is derived from the synthetic promoter, P-Zm.GSP990.nno:2.

SEQ ID NO:19 is a DNA sequence of a 3′ UTR, T-Sb.Nltp4-1:1:2 derivedfrom the NLTP4 (non-specific lipid-transfer protein 4) gene from Sorghumbicolor.

SEQ ID NO:20 is a synthetic coding sequence optimized for plantexpression for β-glucuronidase (GUS) with a processable intron derivedfrom the potato light-inducible tissue-specific ST-LS1 gene (GenbankAccession: X04753).

SEQ ID NO:21 is a DNA sequence of the EXP, EXP-CaMV.35S comprising the35S promoter and leader derived from the Cauliflower mosaic virus.

SEQ ID NO:22 is a DNA sequence of the intron, I-Zm.DnaK:1 derived fromthe heat shock protein 70 (Hsp70) gene (DnaK) from Zea mays.

SEQ ID NO:23 is a DNA sequence of the 3′ UTR, T-Os.LTP:1 derived fromthe Lipid Transfer Protein-like gene (LTP) from Oryza sativa.

SEQ ID NO:24 is a coding sequence for β-glucuronidase (GUS) with aprocessable intron derived from the potato light-inducibletissue-specific ST-LS1 gene (Genbank Accession: X04753).

SEQ ID NO:25 is a coding sequence for the NanoLuc® luciferasefluorescent protein (Promega, Madison, Wis. 53711), Nluc which wasengineered by directed evolution from a deep-sea shrimp (Oplophorusgacilirostris) luciferase.

SEQ ID NO:26 is a DNA sequence of a synthetic 3′ UTR, T-Zm.GST43.nno:1.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides synthetic regulatory elements havinggene-regulatory activity in plants. The nucleotide sequences of thesesynthetic regulatory elements are provided as SEQ ID NOs:1-18 and SEQ IDNO:26. These synthetic regulatory elements are capable of affecting theexpression of an operably linked transcribable DNA molecule in planttissues, and therefore regulating gene expression of an operably linkedtransgene in transgenic plants. The invention also provides novelendogenous regulatory elements having gene-regulatory activity in plantsand provided as SEQ ID NO:19. The invention also provides methods ofmodifying, producing, and using recombinant DNA molecules which containthe provided synthetic and endogenous regulatory elements. The inventionalso provides compositions that include transgenic plant cells, plants,plant parts, and seeds containing the recombinant DNA molecules of theinvention, and methods for preparing and using the same.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

DNA Molecules

As used herein, the term “DNA” or “DNA molecule” refers to adouble-stranded DNA molecule of genomic or synthetic origin, i.e., apolymer of deoxyribonucleotide bases or a DNA molecule, read from the 5′(upstream) end to the 3′ (downstream) end. As used herein, the term “DNAsequence” refers to the nucleotide sequence of a DNA molecule. Thenomenclature used herein corresponds to that of Title 37 of the UnitedStates Code of Federal Regulations § 1.822, and set forth in the tablesin WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

As used herein, a “recombinant DNA molecule” is a DNA moleculecomprising a combination of DNA molecules that would not naturally occurtogether without human intervention. For instance, a recombinant DNAmolecule may be a DNA molecule that is comprised of at least two DNAmolecules heterologous with respect to each other, a DNA molecule thatcomprises a DNA sequence that deviates from DNA sequences that exist innature, a DNA molecule that comprises a synthetic DNA sequence or a DNAmolecule that has been incorporated into a host cell's DNA by genetictransformation or gene editing.

As used herein, a “synthetic nucleotide sequence” or “artificialnucleotide sequence” is a nucleotide sequence that is not known to occurin nature or that is not naturally occurring. The gene-regulatoryelements of the present invention comprise synthetic nucleotidesequences. Preferably, synthetic nucleotide sequences share little or noextended homology to natural sequences. Extended homology in thiscontext generally refers to 100% sequence identity extending beyondabout 25 nucleotides of contiguous sequence.

Reference in this application to an “isolated DNA molecule”, or anequivalent term or phrase, is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. Similarly, a nucleotide sequence encoding an insecticidal proteinor any naturally occurring insecticidal variant of that protein would bean isolated nucleotide sequence so long as the nucleotide sequence wasnot within the DNA of the bacterium from which the sequence encoding theprotein is naturally found. A synthetic nucleotide sequence encoding theamino acid sequence of the naturally occurring insecticidal proteinwould be considered to be isolated for the purposes of this disclosure.For the purposes of this disclosure, any transgenic nucleotide sequence,i.e., the nucleotide sequence of the DNA inserted into the genome of thecells of a plant or bacterium, or present in an extrachromosomal vector,would be considered to be an isolated nucleotide sequence whether it ispresent within the plasmid or similar structure used to transform thecells, within the genome of the plant or bacterium, or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the plant or bacterium.

As used herein, the term “sequence identity” refers to the extent towhich two optimally aligned polynucleotide sequences or two optimallyaligned polypeptide sequences are identical. An optimal sequencealignment is created by manually aligning two sequences, e.g., areference sequence and another sequence, to maximize the number ofnucleotide matches in the sequence alignment with appropriate internalnucleotide insertions, deletions, or gaps. As used herein, the term“reference sequence” refers to a DNA sequence provided as SEQ IDNOs:1-19 and SEQ ID NO:26.

As used herein, the term “percent sequence identity” or “percentidentity” or “% identity” is the identity fraction multiplied by 100.The “identity fraction” for a sequence optimally aligned with areference sequence is the number of nucleotide matches in the optimalalignment, divided by the total number of nucleotides in the referencesequence, e.g., the total number of nucleotides in the full length ofthe entire reference sequence. Thus, one embodiment of the inventionprovides a DNA molecule comprising a sequence that, when optimallyaligned to a reference sequence, provided herein as SEQ ID NOs:1-19 andSEQ ID NO:26, has at least about 85 percent identity, at least about 86percent identity, at least about 87 percent identity, at least about 88percent identity, at least about 89 percent identity, at least about 90percent identity, at least about 91 percent identity, at least about 92percent identity, at least about 93 percent identity, at least about 94percent identity, at least about 95 percent identity, at least about 96percent identity, at least about 97 percent identity, at least about 98percent identity, at least about 99 percent identity, or at least about100 percent identity to the reference sequence. DNA molecules having apercent sequence identity with reference molecule may exhibit theactivity of the reference sequence.

Regulatory Elements

Regulatory elements such as promoters, leaders (also known as 5′ UTRs),enhancers, introns, and transcription termination regions (or 3′ UTRs)play an integral part in the overall expression of genes in livingcells. The term “regulatory element,” as used herein, refers to a DNAmolecule having gene-regulatory activity. The term “gene-regulatoryactivity,” as used herein, refers to the ability to affect theexpression of an operably linked transcribable DNA molecule, forinstance by affecting the transcription and/or translation of theoperably linked transcribable DNA molecule. Regulatory elements, such aspromoters, leaders, enhancers, introns and 3′ UTRs that function inplants are useful for modifying plant phenotypes through geneticengineering.

As used herein, a “regulatory expression element group” or “EXP”sequence may refer to a group of operably linked regulatory elements,such as enhancers, promoters, leaders, and introns. For example, aregulatory expression element group may be comprised, for instance, of apromoter operably linked 5′ to a leader sequence. EXP's useful inpracticing the present invention include SEQ ID NOs:1, 4, 6, 9, 11, 15,and 16.

Regulatory elements may be characterized by their gene expressionpattern, e.g., positive and/or negative effects such as constitutiveexpression or temporal, spatial, developmental, tissue, environmental,physiological, pathological, cell cycle, and/or chemically responsiveexpression, and any combination thereof, as well as by quantitative orqualitative indications. As used herein, a “gene expression pattern” isany pattern of transcription of an operably linked DNA molecule into atranscribed RNA molecule. The transcribed RNA molecule may be translatedto produce a protein molecule or may provide an antisense or otherregulatory RNA molecule, such as a double-stranded RNA (dsRNA), atransfer RNA (tRNA), a ribosomal RNA (rRNA), a microRNA (miRNA), a smallinterfering RNA (siRNA), and the like.

As used herein, the term “protein expression” is any pattern oftranslation of a transcribed RNA molecule into a protein molecule.Protein expression may be characterized by its temporal, spatial,developmental, or morphological qualities, as well as by quantitative orqualitative indications.

A promoter is useful as a regulatory element for modulating theexpression of an operably linked transcribable DNA molecule. As usedherein, the term “promoter” refers generally to a DNA molecule that isinvolved in recognition and binding of RNA polymerase II and otherproteins, such as trans-acting transcription factors, to initiatetranscription. A promoter may be initially isolated from the 5′untranslated region (5′ UTR) of a genomic copy of a gene. Alternately,promoters may be synthetically produced or manipulated DNA molecules.Promoters may also be chimeric. Chimeric promoters are produced throughthe fusion of two or more heterologous DNA molecules. Promoters usefulin practicing the present invention include promoter elements providedas SEQ ID NOs:2 and 7, or comprised within any of SEQ ID NOs:1, 4, 6, 9,11, 15, and 16, or fragments or variants thereof. In specificembodiments of the invention, the claimed DNA molecules and any variantsor derivatives thereof as described herein, are further defined ascomprising promoter activity, i.e., are capable of acting as a promoterin a host cell, such as in a transgenic plant. In still further specificembodiments, a fragment may be defined as exhibiting promoter activitypossessed by the starting promoter molecule from which it is derived, ora fragment may comprise a “minimal promoter” which provides a basallevel of transcription and is comprised of a TATA box or equivalent DNAsequence for recognition and binding of the RNA polymerase II complexfor initiation of transcription.

In one embodiment, fragments of an EXP sequence or a promoter sequencedisclosed herein are provided. Promoter fragments may comprise promoteractivity, as described above, and may be useful alone or in combinationwith other promoters and promoter fragments, such as in constructingchimeric promoters, or in combination with other expression elements andexpression element fragments. In specific embodiments, fragments of apromoter are provided comprising at least about 50, at least about 75,at least about 95, at least about 100, at least about 125, at leastabout 150, at least about 175, at least about 200, at least about 225,at least about 250, at least about 275, at least about 300, at leastabout 500, at least about 600, at least about 700, at least about 750,at least about 800, at least about 900, or at least about 1000contiguous nucleotides, or longer, of a DNA molecule having promoteractivity as disclosed herein. Methods for producing such fragments froma starting promoter molecule are well known in the art.

In further embodiments, fragments of enhancer or intron sequencesdisclosed herein are provided. Enhancer or intron fragments may comprisethe activity of the base molecule from which they were derived, and maybe useful alone or in combination with other regulatory elementsincluding promoters, leaders, other enhancers, other introns, orfragments thereof. In specific embodiments, fragments of an enhancer orintron are provided comprising at least about 50, at least about 75, atleast about 95, at least about 100, at least about 125, at least about150, at least about 175, at least about 200, at least about 225, atleast about 250, at least about 275, at least about 300, at least about500, at least about 600, at least about 700, at least about 750, atleast about 800, at least about 900, or at least about 1000 contiguousnucleotides, or longer, of a DNA molecule having enhancer or intronactivity as disclosed herein. Methods for producing such fragments froma starting molecule are well known in the art.

In other embodiments, fragments of 3′ UTR sequences disclosed herein areprovided. 3′ UTR fragments may comprise the activity of the base 3′ UTRmolecule from which they were derived, and may be useful alone or incombination with other regulatory elements including promoters, leaders,introns, or fragments thereof. In specific embodiments, fragments of anintron are provided comprising at least about 50, at least about 75, atleast about 95, at least about 100, at least about 125, at least about150, at least about 175, at least about 200, at least about 225, atleast about 250, at least about 275, at least about 300, at least about500, at least about 600, at least about 700, at least about 750, atleast about 800, at least about 900, or at least about 1000 contiguousnucleotides, or longer, of a DNA molecule having 3′ UTR activity asdisclosed herein. Methods for producing such fragments from a starting3′ UTR molecule are well known in the art.

Compositions derived from any of the promoter elements provided as SEQID NOs:2 and 7, or comprised within any of SEQ ID NOs:1, 4, 6, 9, 11,15, and 16 such as internal or 5′ deletions, for example, can beproduced using methods known in the art to improve or alter expression,including by removing elements that have either positive or negativeeffects on expression; duplicating elements that have positive ornegative effects on expression; and/or duplicating or removing elementsthat have tissue- or cell-specific effects on expression. Compositionsderived from any of the promoter elements provided as SEQ ID NOs:2 and7, or comprised within any of SEQ ID NOs:1, 4, 6, 9, 11, 15, and 16,comprised of 3′ deletions in which the TATA box element or equivalentsequence thereof and downstream sequence is removed can be used, forexample, to make enhancer elements. Further deletions can be made toremove any elements that have positive or negative; tissue-specific;cell-specific; or timing-specific (such as, but not limited to,circadian rhythm) effects on expression. Any of the promoter elementsprovided as SEQ ID NOs:2 and 7, or comprised within any of SEQ ID NOs:1,4, 6, 9, 11, 15, and 16 and fragments or enhancers derived therefrom canbe used to make chimeric transcriptional regulatory elementcompositions.

In accordance with the invention, a promoter or promoter fragment may beanalyzed for the presence of known promoter elements, i.e., DNA sequencecharacteristics, such as a TATA box and other known transcription factorbinding site motifs. Identification of such known promoter elements maybe used by one of skill in the art to design variants of the promoterhaving a similar expression pattern to the original promoter.

As used herein, the term “leader” refers to a DNA molecule isolated fromthe untranslated 5′ region (5′ UTR) a gene and defined generally as anucleotide segment between the transcription start site (TSS) and theprotein coding sequence start site. Alternately, leaders may besynthetically produced or manipulated DNA elements. A leader can be usedas a 5′ regulatory element for modulating expression of an operablylinked transcribable DNA molecule. Leader molecules may be used with aheterologous promoter or with their native promoter. Leaders useful inpracticing the present invention include SEQ ID NOs:3 and 8; or any ofthe leader elements comprised within any of SEQ ID NOs:1, 4, 6, 9, 11,15, and 16 or fragments or variants thereof. In specific embodiments,such DNA sequences may be defined as being capable of acting as a leaderin a host cell, including, for example, a transgenic plant cell. In oneembodiment, such sequences are decoded as comprising leader activity.

The leader sequences (also referred to as 5′ UTRs) presented as SEQ IDNOs:3 and 8 or any of the leader elements comprised within any of SEQ IDNOs:1, 4, 6, 9, 11, 15, and 16 may be comprised of regulatory elements,or may adopt secondary structures that can have an effect ontranscription or translation of an operably linked transcribable DNAmolecule. The leader sequences presented as SEQ ID NOs:3 and 8 or any ofthe leader elements comprised within any of SEQ ID NOs:1, 4, 6, 9, 11,15, and 16 can be used in accordance with the invention to make chimericregulatory elements that affect transcription or translation of a anoperably linked transcribable DNA molecule.

As used herein, the term “intron” refers to a DNA molecule that may beisolated or identified from a gene and may be defined generally as aregion spliced out during messenger RNA (mRNA) processing prior totranslation. Alternately, an intron may be a synthetically produced ormanipulated DNA element. An intron may contain enhancer elements thateffect the transcription of operably linked genes. An intron may be usedas a regulatory element for modulating expression of an operably linkedtranscribable DNA molecule. A construct may comprise an intron, and theintron may or may not be heterologous with respect to the transcribableDNA molecule. Examples of introns in the art include the rice actinintron and the corn HSP70 intron.

In plants, the inclusion of some introns in gene constructs leads toincreased mRNA and protein accumulation relative to constructs lackingthe intron. This effect has been termed “intron mediated enhancement”(IME) of gene expression. Introns known to stimulate expression inplants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, andUbi1), in rice genes (e.g., tpi) and in dicotyledonous plant genes likethose from petunia (e.g., rbcS), potato (e.g., st-ls1) and fromArabidopsis thaliana (e.g., ubq3 and pati). It has been shown thatdeletions or mutations within the splice sites of an intron reduce geneexpression, indicating that splicing might be needed for IME. However,IME in dicotyledonous plants has been shown by point mutations withinthe splice sites of the pati gene from A. thaliana. Multiple uses of thesame intron in one plant has been shown to exhibit disadvantages. Inthose cases, it is necessary to have a collection of basic controlelements for the construction of appropriate recombinant DNA elements.Exemplary introns useful in practicing the present invention arepresented as SEQ ID NOs:5, 10, and 12.

As used herein, the terms “3′ transcription termination molecule,” “3′untranslated region” or “3′ UTR” refer to a DNA molecule that is usedduring transcription to the untranslated region of the 3′ portion of anmRNA molecule. The 3′ untranslated region of an mRNA molecule may begenerated by specific cleavage and 3′ polyadenylation, also known as apolyA tail. A 3′ UTR may be operably linked to and located downstream ofa transcribable DNA molecule and may include a polyadenylation signaland other regulatory signals capable of affecting transcription, mRNAprocessing, or gene expression. PolyA tails are thought to function inmRNA stability and in initiation of translation. Examples of 3′transcription termination molecules in the art are the nopaline synthase3′ region, wheat hsp17 3′ region, pea rubisco small subunit 3′ region,cotton E6 3′ region, and the coixin 3′ UTR.

3′ UTRs typically find beneficial use for the recombinant expression ofspecific DNA molecules. A weak 3′ UTR has the potential to generateread-through, which may affect the expression of the DNA moleculelocated in the neighboring expression cassettes. Appropriate control oftranscription termination can prevent read-through into DNA sequences(e.g., other expression cassettes) localized downstream and can furtherallow efficient recycling of RNA polymerase to improve gene expression.Efficient termination of transcription (release of RNA Polymerase IIfrom the DNA) is prerequisite for re-initiation of transcription andthereby directly affects the overall transcript level. Subsequent totranscription termination, the mature mRNA is released from the site ofsynthesis and template transported to the cytoplasm. Eukaryotic mRNAsare accumulated as poly(A) forms in vivo, making it difficult to detecttranscriptional termination sites by conventional methods. However,prediction of functional and efficient 3′ UTRs by bioinformatics methodsis difficult in that there are no conserved DNA sequences that wouldallow easy prediction of an effective 3′ UTR.

From a practical standpoint, it is typically beneficial that a 3′ UTRused in an expression cassette possesses the following characteristics.First, the 3′ UTR should be able to efficiently and effectivelyterminate transcription of the transgene and prevent read-through of thetranscript into any neighboring DNA sequence, which can be comprised ofanother expression cassette as in the case of multiple expressioncassettes residing in one transfer DNA (T-DNA), or the neighboringchromosomal DNA into which the T-DNA has inserted. Second, the 3′ UTRshould not cause a reduction in the transcriptional activity imparted bythe promoter, leader, enhancers, and introns that are used to driveexpression of the DNA molecule. Finally, in plant biotechnology, the 3′UTR is often used for priming of amplification reactions of reversetranscribed RNA extracted from the transformed plant and used to: (1)assess the transcriptional activity or expression of the expressioncassette once integrated into the plant chromosome; (2) assess the copynumber of insertions within the plant DNA; and (3) assess zygosity ofthe resulting seed after breeding. The 3′ UTR is also used inamplification reactions of DNA extracted from the transformed plant tocharacterize the intactness of the inserted cassette. 3′ UTRs useful inpracticing the present invention are presented as SEQ ID NOs:13, 14, 19,and 26.

As used herein, the term “enhancer” or “enhancer element” refers to acis-acting regulatory element, a.k.a. cis-element, which confers anaspect of the overall expression pattern, but is usually insufficientalone to drive transcription, of an operably linked transcribable DNAmolecule. Unlike promoters, enhancer elements do not usually include atranscription start site (TSS) or TATA box or equivalent DNA sequence. Apromoter or promoter fragment may naturally comprise one or moreenhancer elements that affect the transcription of an operably linkedDNA sequence. An enhancer element may also be fused to a promoter toproduce a chimeric promoter cis-element, which confers an aspect of theoverall modulation of gene expression.

Many promoter enhancer elements are believed to bind DNA-bindingproteins and/or affect DNA topology, producing local conformations thatselectively allow or restrict access of RNA polymerase to the DNAtemplate or that facilitate selective opening of the double helix at thesite of transcriptional initiation. An enhancer element may function tobind transcription factors that regulate transcription. Some enhancerelements bind more than one transcription factor, and transcriptionfactors may interact with different affinities with more than oneenhancer domain. Enhancer elements can be identified by a number oftechniques, including deletion analysis, i.e., deleting one or morenucleotides from the 5′ end or internal to a promoter; DNA bindingprotein analysis using DNase I footprinting, methylation interference,electrophoresis mobility-shift assays, in vivo genomic footprinting byligation-mediated polymerase chain reaction (PCR), and otherconventional assays or by DNA sequence similarity analysis using knowncis-element motifs or enhancer elements as a target sequence or targetmotif with conventional DNA sequence comparison methods, such as BLAST.The fine structure of an enhancer domain can be further studied bymutagenesis (or substitution) of one or more nucleotides or by otherconventional methods known in the art. Enhancer elements can be obtainedby chemical synthesis or by isolation from regulatory elements thatinclude such elements, and they can be synthesized with additionalflanking nucleotides that contain useful restriction enzyme sites tofacilitate subsequence manipulation. Thus, the design, construction, anduse of enhancer elements according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the invention. Exemplary enhancers useful inpracticing this invention are presented as SEQ ID NOs:17 and 18.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither the first nor the second DNA molecule would normally be found inthat configuration, i.e. fused to the other. The chimeric DNA moleculeis thus a new DNA molecule not otherwise normally found in nature. Asused herein, the term “chimeric promoter” refers to a promoter producedthrough such manipulation of DNA molecules. A chimeric promoter maycombine two or more DNA fragments for example, the fusion of a promoterto an enhancer element. Thus, the design, construction, and use ofchimeric promoters according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the present invention.

Chimeric regulatory elements can be designed to comprise variousconstituent elements which may be operatively linked by various methodsknown in the art, such as restriction enzyme digestion and ligation,ligation independent cloning, modular assembly of PCR products duringamplification, or direct chemical synthesis of the regulatory element,as well as other methods known in the art. The resulting variouschimeric regulatory elements can be comprised of the same, or variantsof the same, constituent elements but differ in the DNA sequence or DNAsequences that comprise the linking DNA sequence or sequences that allowthe constituent parts to be operatively linked. In the invention, theDNA sequences provided as SEQ ID NOs:1-19 and SEQ ID NO:26 may provideregulatory element reference sequences, wherein the constituent elementsthat comprise the reference sequence may be joined by methods known inthe art and may comprise substitutions, deletions, and/or insertions ofone or more nucleotides or mutations that naturally occur in bacterialand plant cell transformation.

As used herein, the term “variant” refers to a second DNA molecule, suchas a regulatory element, that is in composition similar, but notidentical to, a first DNA molecule, and wherein the second DNA moleculestill maintains the general functionality, i.e. the same or similarexpression pattern, for instance through more or less equivalenttranscriptional activity, of the first DNA molecule. A variant may be ashorter or truncated version of the first DNA molecule or an alteredversion of the sequence of the first DNA molecule, such as one withdifferent restriction enzyme sites and/or internal deletions,substitutions, or insertions. A “variant” can also encompass aregulatory element having a nucleotide sequence comprising asubstitution, deletion, or insertion of one or more nucleotides of areference sequence, wherein the derivative regulatory element has moreor less or equivalent transcriptional or translational activity than thecorresponding parent regulatory molecule. Regulatory element “variants”will also encompass variants arising from mutations that naturally occurin bacterial and plant cell transformation. In the present invention, apolynucleotide sequence provided as SEQ ID NOs:1-19 and SEQ ID NO:26 maybe used to create variants that are similar in composition, but notidentical to, the DNA sequence of the original regulatory element, whilestill maintaining the general functionality, i.e., the same or similarexpression pattern, of the original regulatory element. Production ofsuch variants of the invention is well within the ordinary skill of theart in light of the disclosure and is encompassed within the scope ofthe invention.

The efficacy of the modifications, duplications, or deletions describedherein on the desired expression aspects of a particular transgene maybe tested empirically in stable and transient plant assays, such asthose described in the working examples herein, so as to validate theresults, which may vary depending upon the changes made and the goal ofthe change in the starting DNA molecule.

Constructs

As used herein, the term “construct” means any recombinant DNA moleculesuch as a plasmid, cosmid, virus, phage, or linear or circular DNA orRNA molecule, derived from any source, capable of genomic integration orautonomous replication, comprising a DNA molecule where at least one DNAmolecule has been linked to another DNA molecule in a functionallyoperative manner, i.e. operably linked. As used herein, the term“vector” means any construct that may be used for the purpose oftransformation, i.e., the introduction of heterologous DNA or RNA into ahost cell. A construct typically includes one or more expressioncassettes. As used herein, an “expression cassette” refers to a DNAmolecule comprising at least a transcribable DNA molecule operablylinked to one or more regulatory elements, typically at least a promoterand a 3′ UTR.

As used herein, the term “operably linked” refers to a first DNAmolecule joined to a second DNA molecule, wherein the first and secondDNA molecules are so arranged that the first DNA molecule affects thefunction of the second DNA molecule. The two DNA molecules may or maynot be part of a single contiguous DNA molecule and may or may not beadjacent. For example, a promoter is operably linked to a transcribableDNA molecule if the promoter modulates transcription of thetranscribable DNA molecule of interest in a cell. A leader, for example,is operably linked to DNA sequence when it is capable of affecting thetranscription or translation of the DNA sequence.

The constructs of the invention may be provided, in one embodiment, asdouble tumor-inducing (Ti) plasmid border constructs that have the rightborder (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of theTi plasmid isolated from Agrobacterium tumefaciens comprising a T-DNAthat, along with transfer molecules provided by the A. tumefacienscells, permit the integration of the T-DNA into the genome of a plantcell (see, e.g., U.S. Pat. No. 6,603,061). The constructs may alsocontain the plasmid backbone DNA segments that provide replicationfunction and antibiotic selection in bacterial cells, e.g., anEscherichia coli origin of replication such as ori322, a broad hostrange origin of replication such as oriV or oriRi, and a coding regionfor a selectable marker such as Spec/Strp that encodes for Tn7aminoglycoside adenyltransferase (aadA) conferring resistance tospectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectablemarker gene. For plant transformation, the host bacterial strain isoften A. tumefaciens ABI, C58, or LBA4404, however other strains knownto those skilled in the art of plant transformation can function in theinvention.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the transcribable DNA molecule istranscribed into a functional mRNA molecule that is translated andexpressed as a protein. For the practice of the invention, conventionalcompositions and methods for preparing and using constructs and hostcells are well known to one skilled in the art. Typical vectors usefulfor expression of nucleic acids in higher plants are well known in theart and include vectors derived from the Ti plasmid of Agrobacteriumtumefaciens and the pCaMVCN transfer control vector.

Various regulatory elements may be included in a construct, includingany of those provided herein. Any such regulatory elements may beprovided in combination with other regulatory elements. Suchcombinations can be designed or modified to produce desirable regulatoryfeatures. In one embodiment, constructs of the invention comprise atleast one regulatory element operably linked to a transcribable DNAmolecule operably linked to a 3′ UTR.

Constructs of the invention may include any promoter or leader providedherein or known in the art. For example, a promoter of the invention maybe operably linked to a heterologous non-translated 5′ leader such asone derived from a heat shock protein gene. Alternatively, a leader ofthe invention may be operably linked to a heterologous promoter such asthe Cauliflower Mosaic Virus 35S transcript promoter.

Expression cassettes may also include a transit peptide coding sequencethat encodes a peptide that is useful for sub-cellular targeting of anoperably linked protein, particularly to a chloroplast, leucoplast, orother plastid organelle; mitochondria; peroxisome; vacuole; or anextracellular location. Many chloroplast-localized proteins areexpressed from nuclear genes as precursors and are targeted to thechloroplast by a chloroplast transit peptide (CTP). Examples of suchisolated chloroplast proteins include, but are not limited to, thoseassociated with the small subunit (SSU) of ribulose-1,5-bisphosphatecarboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvestingcomplex protein I and protein II, thioredoxin F, and enolpyruvylshikimate phosphate synthase (EPSPS). Chloroplast transit peptides aredescribed, for example, in U.S. Pat. No. 7,193,133. It has beendemonstrated that non-chloroplast proteins may be targeted to thechloroplast by the expression of a heterologous CTP operably linked tothe transgene encoding a non-chloroplast proteins.

Transcribable DNA Molecules

As used herein, the term “transcribable DNA molecule” refers to any DNAmolecule capable of being transcribed into an RNA molecule, including,but not limited to, those having protein coding sequences and thoseproducing RNA molecules having sequences useful for gene suppression.The type of DNA molecule can include, but is not limited to, a DNAmolecule from the same plant, a DNA molecule from another plant, a DNAmolecule from a different organism, or a synthetic DNA molecule, such asa DNA molecule containing an antisense message of a gene, or a DNAmolecule encoding an artificial, synthetic, or otherwise modifiedversion of a transgene. Exemplary transcribable DNA molecules forincorporation into constructs of the invention include, e.g., DNAmolecules or genes from a species other than the species into which theDNA molecule is incorporated or genes that originate from, or arepresent in, the same species, but are incorporated into recipient cellsby genetic engineering methods rather than classical breedingtechniques.

A “transgene” refers to a transcribable DNA molecule heterologous to ahost cell at least with respect to its location in the host cell genomeand/or a transcribable DNA molecule artificially incorporated into ahost cell's genome in the current or any prior generation of the cell.

A regulatory element, such as a promoter of the invention, may beoperably linked to a transcribable DNA molecule that is heterologouswith respect to the regulatory element. As used herein, the term“heterologous” refers to the combination of two or more DNA moleculeswhen such a combination is not normally found in nature. For example,the two DNA molecules may be derived from different species and/or thetwo DNA molecules may be derived from different genes, e.g., differentgenes from the same species or the same genes from different species. Aregulatory element is thus heterologous with respect to an operablylinked transcribable DNA molecule if such a combination is not normallyfound in nature, i.e., the transcribable DNA molecule does not naturallyoccur operably linked to the regulatory element.

The transcribable DNA molecule may generally be any DNA molecule forwhich expression of a transcript is desired. Such expression of atranscript may result in translation of the resulting mRNA molecule, andthus protein expression. Alternatively, for example, a transcribable DNAmolecule may be designed to ultimately cause decreased expression of aspecific gene or protein. In one embodiment, this may be accomplished byusing a transcribable DNA molecule that is oriented in the antisensedirection. One of ordinary skill in the art is familiar with using suchantisense technology. Any gene may be negatively regulated in thismanner, and, in one embodiment, a transcribable DNA molecule may bedesigned for suppression of a specific gene through expression of adsRNA, siRNA or miRNA molecule.

Thus, one embodiment of the invention is a recombinant DNA moleculecomprising a regulatory element of the invention, such as those providedas SEQ ID NOs:1-19 and SEQ ID NO:26, operably linked to a heterologoustranscribable DNA molecule so as to modulate transcription of thetranscribable DNA molecule at a desired level or in a desired patternwhen the construct is integrated in the genome of a transgenic plantcell. In one embodiment, the transcribable DNA molecule comprises aprotein-coding region of a gene and in another embodiment thetranscribable DNA molecule comprises an antisense region of a gene.

Genes of Agronomic Interest

A transcribable DNA molecule may be a gene of agronomic interest. Asused herein, the term “gene of agronomic interest” refers to atranscribable DNA molecule that, when expressed in a particular planttissue, cell, or cell type, confers a desirable characteristic. Theproduct of a gene of agronomic interest may act within the plant inorder to cause an effect upon the plant morphology, physiology, growth,development, yield, grain composition, nutritional profile, disease orpest resistance, and/or environmental or chemical tolerance or may actas a pesticidal agent in the diet of a pest that feeds on the plant. Inone embodiment of the invention, a regulatory element of the inventionis incorporated into a construct such that the regulatory element isoperably linked to a transcribable DNA molecule that is a gene ofagronomic interest. In a transgenic plant containing such a construct,the expression of the gene of agronomic interest can confer a beneficialagronomic trait. A beneficial agronomic trait may include, for example,but is not limited to, herbicide tolerance, insect control, modifiedyield, disease resistance, pathogen resistance, modified plant growthand development, modified starch content, modified oil content, modifiedfatty acid content, modified protein content, modified fruit ripening,enhanced animal and human nutrition, biopolymer productions,environmental stress resistance, pharmaceutical peptides, improvedprocessing qualities, improved flavor, hybrid seed production utility,improved fiber production, and desirable biofuel production.

Non-limiting examples of genes of agronomic interest known in the artinclude those for herbicide resistance (U.S. Pat. Nos. 6,803,501;6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425;5,633,435; and 5,463,175), increased yield (U.S. Pat. Nos. USRE38,446;6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330;6,372,211; 6,235,971; 6,222,098; and 5,716,837), insect control (U.S.Pat. Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497;6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109;6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378;6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615;6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597;6,023,013; 5,959,091; 5,942,664; 5,942,658, 5,880,275; 5,763,245; and5,763,241), fungal disease resistance (U.S. Pat. Nos. 6,653,280;6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696;6,121,436; 6,316,407; and 6,506,962), virus resistance (U.S. Pat. Nos.6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730),nematode resistance (U.S. Pat. No. 6,228,992), bacterial diseaseresistance (U.S. Pat. No. 5,516,671), plant growth and development (U.S.Pat. Nos. 6,723,897 and 6,518,488), starch production (U.S. Pat. Nos.6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oilsproduction (U.S. Pat. Nos. 6,444,876; 6,426,447; and 6,380,462), highoil production (U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; and6,476,295), modified fatty acid content (U.S. Pat. Nos. 6,828,475;6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767;6,537,750; 6,489,461; and 6,459,018), high protein production (U.S. Pat.No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhancedanimal and human nutrition (U.S. Pat. Nos. 6,723,837; 6,653,530;6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Pat. Nos.USRE37,543; 6,228,623; and U.S. Pat. Nos. 5,958,745, and 6,946,588),environmental stress resistance (U.S. Pat. No. 6,072,103),pharmaceutical peptides and secretable peptides (U.S. Pat. Nos.6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processingtraits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No.6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzymeproduction (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No.6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seedproduction (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. Nos.6,576,818; 6,271,443; 5,981,834; and 5,869,720) and biofuel production(U.S. Pat. No. 5,998,700).

Alternatively, a gene of agronomic interest can affect the abovementioned plant characteristics or phenotypes by encoding a RNA moleculethat causes the targeted modulation of gene expression of an endogenousgene, for example by antisense (see, e.g. U.S. Pat. No. 5,107,065);inhibitory RNA (“RNAi,” including modulation of gene expression bymiRNA-, siRNA-, trans-acting siRNA-, and phased sRNA-mediatedmechanisms, e.g., as described in published applications U.S.2006/0200878 and U.S. 2008/0066206, and in U.S. patent application Ser.No. 11/974,469); or cosuppression-mediated mechanisms. The RNA couldalso be a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see,e.g., U.S. 2006/0200878) engineered to cleave a desired endogenous mRNAproduct. Methods are known in the art for constructing and introducingconstructs into a cell in such a manner that the transcribable DNAmolecule is transcribed into a molecule that is capable of causing genesuppression.

Selectable Markers

Selectable marker transgenes may also be used with the regulatoryelements of the invention. As used herein the term “selectable markertransgene” refers to any transcribable DNA molecule whose expression ina transgenic plant, tissue or cell, or lack thereof, can be screened foror scored in some way. Selectable marker genes, and their associatedselection and screening techniques, for use in the practice of theinvention are known in the art and include, but are not limited to,transcribable DNA molecules encoding β-glucuronidase (GUS), greenfluorescent protein (GFP), proteins that confer antibiotic resistance,and proteins that confer herbicide tolerance. Examples of selectablemarker transgenes is provided as SEQ ID NOs:20 and 24.

Cell Transformation

The invention is also directed to a method of producing transformedcells and plants that comprise one or more regulatory elements operablylinked to a transcribable DNA molecule.

The term “transformation” refers to the introduction of a DNA moleculeinto a recipient host. As used herein, the term “host” refers tobacteria, fungi, or plants, including any cells, tissues, organs, orprogeny of the bacteria, fungi, or plants. Plant tissues and cells ofparticular interest include protoplasts, calli, roots, tubers, seeds,stems, leaves, seedlings, embryos, and pollen.

As used herein, the term “transformed” refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is inherited by subsequent progeny. A“transgenic” or “transformed” cell or organism may also include progenyof the cell or organism and progeny produced from a breeding programemploying such a transgenic organism as a parent in a cross andexhibiting an altered phenotype resulting from the presence of a foreignDNA molecule. The introduced DNA molecule may also be transientlyintroduced into the recipient cell such that the introduced DNA moleculeis not inherited by subsequent progeny. The term “transgenic” refers toa bacterium, fungus, or plant containing one or more heterologous DNAmolecules.

There are many methods well known to those of skill in the art forintroducing DNA molecules into plant cells. The process generallycomprises the steps of selecting a suitable host cell, transforming thehost cell with a vector, and obtaining the transformed host cell.Methods and materials for transforming plant cells by introducing aplant construct into a plant genome in the practice of this inventioncan include any of the well-known and demonstrated methods. Suitablemethods include, but are not limited to, bacterial infection (e.g.,Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., byPEG-mediated transformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, and accelerationof DNA coated particles), gene editing (e.g., CRISPR-Cas systems), amongothers.

Host cells may be any cell or organism, such as a plant cell, algalcell, algae, fungal cell, fungi, bacterial cell, or insect cell. Inspecific embodiments, the host cells and transformed cells may includecells from crop plants.

A transgenic plant subsequently may be regenerated from a transgenicplant cell of the invention. Using conventional breeding techniques orself-pollination, seed may be produced from this transgenic plant. Suchseed, and the resulting progeny plant grown from such seed, will containthe recombinant DNA molecule of the invention, and therefore will betransgenic.

Transgenic plants of the invention can be self-pollinated to provideseed for homozygous transgenic plants of the invention (homozygous forthe recombinant DNA molecule) or crossed with non-transgenic plants ordifferent transgenic plants to provide seed for heterozygous transgenicplants of the invention (heterozygous for the recombinant DNA molecule).Both such homozygous and heterozygous transgenic plants are referred toherein as “progeny plants.” Progeny plants are transgenic plantsdescended from the original transgenic plant and containing therecombinant DNA molecule of the invention. Seeds produced using atransgenic plant of the invention can be harvested and used to growgenerations of transgenic plants, i.e., progeny plants of the invention,comprising the construct of this invention and expressing a gene ofagronomic interest. Descriptions of breeding methods that are commonlyused for different crops can be found in one of several reference books,see, e.g., Allard, Principles of Plant Breeding, John Wiley & Sons, NY,U. of CA, Davis, Calif., 50-98 (1960); Simmonds, Principles of CropImprovement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen,Plant breeding Perspectives, Wageningen (ed), Center for AgriculturalPublishing and Documentation (1979); Fehr, Soybeans: Improvement,Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr,Principles of Variety Development, Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376 (1987).

The transformed plants may be analyzed for the presence of the gene orgenes of interest and the expression level and/or profile conferred bythe regulatory elements of the invention. Those of skill in the art areaware of the numerous methods available for the analysis of transformedplants. For example, methods for plant analysis include, but are notlimited to, Southern blots or northern blots, PCR-based approaches,biochemical analyses, phenotypic screening methods, field evaluations,and immunodiagnostic assays. The expression of a transcribable DNAmolecule can be measured using TaqMan® (Applied Biosystems, Foster City,Calif.) reagents and methods as described by the manufacturer and PCRcycle times determined using the TaqMan® Testing Matrix. Alternatively,the Invader® (Third Wave Technologies, Madison, Wis.) reagents andmethods as described by the manufacturer can be used to evaluatetransgene expression.

The invention also provides for parts of a plant of the invention. Plantparts include, but are not limited to, leaves, stems, roots, tubers,seeds, endosperm, ovule, and pollen. Plant parts of the invention may beviable, nonviable, regenerable, and/or non-regenerable. The inventionalso includes and provides transformed plant cells comprising a DNAmolecule of the invention. The transformed or transgenic plant cells ofthe invention include regenerable and/or non-regenerable plant cells.

The invention also provides a commodity product that is produced from atransgenic plant or part thereof containing the recombinant DNA moleculeof the invention. Commodity products of the invention contain adetectable amount of DNA comprising a DNA sequence selected from thegroup consisting of SEQ ID NOs:1-19 and SEQ ID NO:26. As used herein, a“commodity product” refers to any composition or product which iscomprised of material derived from a transgenic plant, seed, plant cell,or plant part containing the recombinant DNA molecule of the invention.Commodity products include but are not limited to processed seeds,grains, plant parts, and meal. A commodity product of the invention willcontain a detectable amount of DNA corresponding to the recombinant DNAmolecule of the invention. Detection of one or more of this DNA in asample may be used for determining the content or the source of thecommodity product. Any standard method of detection for DNA moleculesmay be used, including methods of detection disclosed herein.

The invention may be more readily understood through reference to thefollowing examples, which are provided by way of illustration, and arenot intended to be limiting of the invention, unless specified. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the following examples represent techniques discovered bythe inventors to function well in the practice of the invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention, thereforeall matter set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

EXAMPLES Example 1 Design, Synthesis, and Cloning of the SyntheticRegulatory Elements

Novel synthetic transcriptional regulatory elements are syntheticexpression elements designed through algorithmic methods. Thesecomputationally-designed regulatory elements were chemically synthesizedand cloned to make synthetic regulatory expression element groups(EXPs). Well over 1,000 synthetic regulatory elements were designed andassayed in corn protoplasts and stably transformed corn plants toidentify those synthetic regulatory elements that provided desiredcharacteristics such as protein expression levels and patterns ofexpression. The synthetic elements of the present invention providevarious patterns of constitutive expression useful in driving expressionof many different coding sequences and interfering RNAs of agronomicinterest.

The designed synthetic transcriptional regulatory elements do not haveextended homology to any known nucleic acid sequences that exist innature, yet affect transcription of an operably linked coding sequencein the same manner as naturally occurring promoters, leaders, introns,and 3′ UTRs. The synthetic EXPs and their corresponding syntheticpromoters, leaders, and introns as well as synthetic 3′ UTRs arepresented in Table 1. The synthetic EXPs were cloned using methods knownin the art into binary plant transformation vectors, operably linked toa β-glucuronidase (GUS) coding sequence, and the levels and patterns ofexpression in stably transformed corn plants were evaluated.

Analysis of the regulatory element TSS and intron/exon splice junctionscan be performed using transformed plant tissue. Briefly, the plantswere transformed with the plant expression vectors comprising the clonedDNA fragments operably linked to a heterologous transcribable DNAmolecule. Next, the 5′ RACE System for Rapid Amplification of cDNA Ends,Version 2.0 (Invitrogen, Carlsbad, Calif. 92008), was used to confirmthe regulatory element TSS and intron/exon splice junctions by analyzingthe DNA sequence of the produced mRNA transcripts. The synthetic 3′ UTRswere characterized for their effect on gene expression as well as forproper termination of the transcript.

In addition to the synthetic expression elements, a novel endogenous 3′UTR derived from the Sorghum bicolor non-specific lipid-transfer protein4 gene, T-Sb.Nltp4-1:1:2, is provided herein and is presented as SEQ IDNO:19. T-Sb.Nltp4-1:1:2 was characterized in a similar manner as thesynthetic 3′ UTRs.

TABLE 1 Synthetic transcriptional regulatory expression element groups,promoters, leaders, introns, and 3′ UTRs. Description and/or regulatoryelements of EXP linked in 5′ → 3′ direction Annotation SEQ ID NO: Size(bp) (SEQ ID NOs): EXP-Zm.GSP850 1 500 EXP: P-Zm.GSP850.nno: 4 (SEQ IDNO: 2), L-Zm.GSP850.nno: 3 (SEQ ID NO: 3) P-Zm.GSP850.nno: 4 2 450Promoter L-Zm.GSP850.nno: 3 3 50 Leader EXP-Zm.GSP850.nno +Zm.GSI153.nno: 2 4 1117 EXP: P-Zm.GSP850.nno: 4 (SEQ ID NO: 2),L-Zm.GSP850.nno: 3 (SEQ ID NO: 3), I- Zm.GSI153.nno: 1 (SEQ ID NO: 5)I-Zm.GSI153.nno: 1 5 610 Intron EXP-Zm.GSP990 6 500 EXP:P-Zm.GSP990.nno: 2 (SEQ ID NO: 7), L-Zm.GSP990.nno: 1 (SEQ ID NO: 8)P-Zm.GSP990.nno: 2 7 450 Promoter L-Zm.GSP990.nno: 1 8 50 LeaderEXP-Zm.GSP990.nno + Zm.GSI197.nno: 2 9 1117 EXP: P-Zm.GSP990.nno: 2 (SEQID NO: 7), L-Zm.GSP990.nno: 1 (SEQ ID NO: 8), I- Zm.GSI197.nno: 1 (SEQID NO: 10) I-Zm.GSI197.nno: 1 10 610 Intron EXP-Zm.GSP850.nno +Zm.GSI140.nno: l 11 1117 EXP: P-Zm.GSP850.nno: 4 (SEQ ID NO: 2),L-Zm.GSP850.nno: 3 (SEQ ID NO: 3), I- Zm.GSI140.nno: 1 (SEQ ID NO: 12)I-Zm.GSI140.nno: 1 12 610 Intron T-Zm.GST9.nno: 2 13 300 3′ UTRT-Zm.GST18.nno: 2 14 400 3′ UTR EXP-Zm.GSP850.nno + Zm.DnaK: 1 15 1311EXP: P-Zm.GSP850.nno: 4 (SEQ ID NO: 2), L-Zm.GSP850.nno: 3 (SEQ ID NO:3), I- Zm.DnaK: 1 (SEQ ID NO: 22) EXP-Zm.GSP990.nno + Zm.DnaK: 1 16 1311EXP: P-Zm.GSP990.nno: 2 (SEQ ID NO: 7), L-Zm.GSP990.nno: 1 (SEQ ID NO:8), I- Zm.DnaK: 1 (SEQ ID NO: 22) E-Zm.GSP850 17 418 EnhancerE-Zm.GSP990 18 416 Enhancer T-Zm.GST43.nno: 1 26 300 3′ UTR

Example 2 Analysis of the Synthetic Regulatory Elements Driving GUS inCorn Leaf Protoplasts

Corn leaf protoplasts were transformed with vectors, specificallyexpression vectors containing a test regulatory element drivingexpression of the β-glucuronidase (GUS) transgene. The resultingtransformed corn leaf protoplasts were analyzed for GUS proteinexpression to assess the effect of the selected regulatory elements onexpression.

Corn protoplasts, derived from leaf tissue, were transformed withexpression vectors comprising synthetic expression elements. The leveland pattern of expression of these synthetic expression element vectorsin corn protoplasts was compared to the level and pattern of expressionof expression elements known in the art. Separate experiments wereconducted to assess the activity of the EXP's, EXP-Zm.GSP850 (SEQ IDNO:1) and EXP-Zm.GSP990 (SEQ ID NO:6), the introns I-Zm.GSI153.nno:1(SEQ ID NO:5) and I-Zm.GSI197.nno:1 (SEQ ID NO:10), and the 3′ UTR's,T-Zm.GST9.nno:2 (SEQ ID NO:13) and T-Zm.GST18.nno:2 (SEQ ID NO:14). Theexpression elements were cloned into expression vectors and operablylinked to a GUS coding sequence, GOI-Ec.uidA+St.LS1:1:1 (SEQ ID NO:24),that comprised a processable intron. The control expression vectorscomprised different configurations of known expression elements whichvaried dependent on the type of element being assessed (EXP, Intron, or3′ UTR). A plasmid used in co-transformation of the protoplasts andnormalization of the data was also constructed using methods known inthe art. It comprised a transgene cassette comprised of the EXP,EXP-CaMV.35S (SEQ ID NO:21) operably linked 5′ to a coding sequenceencoding the NanoLuc® luciferase fluorescent protein (Promega, Madison,Wis. 53711), herein referred to as Nluc (SEQ ID NO:25), which wasoperably linked 5′ to a 3′ UTR, T-Os.LTP:1 (SEQ ID NO:23).

Corn leaf protoplasts were transformed using a PEG-based transformationmethod, similar to those known in the art. Protoplast cells weretransformed in a ninety six (96) well format. Twelve (12) micrograms ofthe test vector DNA or control vector DNA, and six (6) micrograms of theNanoLuc® vector DNA were used to transform 3.2×10⁵ protoplasts per well.After transformation, the protoplasts were incubated at 25° C. in thedark for sixteen to twenty hours. Following incubation, the protoplastswere lysed and the lysate was used for measuring luciferase and GUSexpression. To lyse the cells, the cells in the plate were pelletedthrough centrifugation, washed, resuspended in a smaller volume, andtransferred to strip well tubes. The tubes were centrifuged again andsupernatant was aspirated leaving the protoplast cell pellet behind. Thecell pellet was resuspended in QB buffer (100 mM KPO4, pH 7.8; 1 mMEDTA; 1% Triton X-100; 10% Glycerol; 1 mM DTT). The cells were lysed byvigorously pipetting the cells several times, vortexing the tubes, andletting the tubes incubate on ice for five minutes. The lysate was thencentrifuged to pellet the cell debris. The resulting lysate was thentransferred to a clean plate.

Luciferase activity was assayed using the Nano-Glo® Luciferase AssaySubstrate (Promega, Madison, Wis. 53711) in QB buffer. In short, a smallvolume of lysate, QB buffer, and the Nano-Glo® Luciferase AssaySubstrate/QB solution were mixed together in white, ninety six (96) wellplates. Fluorescence was then measured using a PHERAstar® plate reader(BMG LABTECH Inc., Cary, N.C. 27513).

GUS activity was assayed using the fluorogenic substrate4-methyleumbelliferyl-β-D-glucuronide (MUG) in a total reaction volumeof fifty (50) microliters. The reaction product, 4-methlyumbelliferone(4-MU), is maximally fluorescent at high pH, where the hydroxyl group isionized. Addition of a basic solution of sodium carbonate simultaneouslystops the assay and adjusts the pH for quantifying the fluorescentproduct. An aliquot of lysate was mixed with an aliquot of MUG dissolvedin QB buffer and incubated at 37° C. A small aliquot of the lysate/MUGreaction mixture was removed and added to a stop buffer at threedifferent time points: (1) immediately after mixing the lysate/MUGreaction as “Time zero minutes”; (2) twenty minutes; and (3) sixtyminutes. Fluorescence was measured with excitation at 355 nm, emissionat 460 nm using a using a PHERAstar® plate reader (BMG LABTECH Inc.,Cary, N.C. 27513). The level of expression is expressed as “nM MUGhydrolyzed” which is derived from the-in-plate standard curve.

For each plate, each construct is transformed in four (4) to eight (8)wells. An aliquot was taken out of each transformation for the MUG assayand “nM MUG hydrolyzed” was derived from the in-plate-standard curve. Analiquot was also taken out of each transformation for the NanoLuc®reading (NanoLuc® RLU). The mean nM MUG hydrolyzed/NanoLuc® RLU for eachconstruct was normalized with respect to theEXP-CaMV.35S/I-Zm.DnaK:1/T-Os.LTP:1 construct which is set to 100%.

Analysis of GUS Expression in Corn Leaf Protoplasts Driven by theSynthetic EXP, EXP-Zm.GSP850.

Corn leaf protoplast cells were transformed with expression vectors thatwere constructed using methods known in the art comprising expressionelements driving GUS expression. Two (2) test expression vectorscomprised transgene cassettes comprising the synthetic EXP,EXP-Zm.GSP850 (SEQ ID NO:1). The synthetic EXP-Zm.GSP850 is comprised ofa synthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO:2), operably linked5′ to a synthetic leader, L-Zm.GSP850.nno:3 (SEQ ID NO:3). The firsttest vector comprised EXP-Zm.GSP850, operably linked 5′ to a codingsequence encoding GUS (SEQ ID NO:24) which comprised a processableintron, which was operably linked 5′ to the 3′ UTR, T-Os.LTP:1 (SEQ IDNO:23). The second transgene cassette comprised EXP-Zm.GSP850, operablylinked 5′ to the intron I-Zm.DnaK:1 (SEQ ID NO:22), operably linked 5′to the GUS coding sequence, which was operably linked 5′ to the 3′ UTR,T-Os.LTP:1.

Three (3) control expression vectors were also constructed and used totransform corn leaf protoplasts. The first control expression vectorcomprised a promoterless transgene cassette and was comprised of theintron, I-Zm.DnaK:1, operably linked 5′ to the GUS coding sequence,which was operably linked 5′ to the 3′ UTR, T-Os.LTP:1. The secondcontrol vector comprised an intronless transgene cassette and wascomprised of the EXP, EXP-CaMV.35S (SEQ ID NO:21), operably linked 5′ tothe GUS coding sequence, which was operably linked 5′ to the 3′ UTR,T-Os.LTP:1. The third control vector comprised a transgene cassettewhich comprised the EXP, EXP-CaMV.35S, operably linked 5′ to the intronI-Zm.DnaK:1, operably linked 5′ to the GUS coding sequence, which wasoperably linked 5′ to the 3′ UTR, T-Os.LTP:1.

Corn leaf protoplasts were transformed with all five (5) vectors.Transformation and lyses of the protoplast cells were performed asdescribed herein. Luciferase and GUS expression were assayed asdescribed herein. Table 2 shows the Mean GUS expression assayed and isexpressed as a percentage of expression relative to the third controlexpression vector that comprises EXP-CaMV.35S and I-Zm.DnaK:1 drivingGUS.

TABLE 2 Mean percent GUS expression of corn leaf protoplasts transformedwith test and control vectors. Plate ID Promoter Intron Mean SD Reps 39No Promoter I-Zm.DnaK: 1 0.1 0.337 6 54 No Promoter I-Zm.DnaK: 1 0.40.315 8 95 No Promoter I-Zm.DnaK: 1 0.1 0.534 8 103 No PromoterI-Zm.DnaK: 1 0.5 0.149 8 39 EXP-CaMV.35S No Intron 48.3 2.018 6 54EXP-CaMV.35S No Intron 46.5 3.949 8 95 EXP-CaMV.35S No Intron 46.8 4.3698 103 EXP-CaMV.35S No Intron 40 3.333 8 39 EXP-CaMV.35S I-Zm.DnaK: 1 1005.117 6 54 EXP-CaMV.35S I-Zm.DnaK: 1 100 6.465 8 95 EXP-CaMV.35SI-Zm.DnaK: 1 100 18.603 8 103 EXP-CaMV.35S I-Zm.DnaK: 1 100 5.164 8 95EXP-Zm.GSP850 No Intron 14 1.844 8 103 EXP-Zm.GSP850 No Intron 8.4 0.3368 39 EXP-Zm.GSP850 I-Zm.DnaK: 1 19 0.732 4 54 EXP-Zm.GSP850 I-Zm.DnaK: 122 1.954 8

As can be seen in Table 2 above, EXP-Zm.GSP850 (SEQ ID NO:1) was able todrive GUS transgene expression in corn leaf protoplasts when compared tocorn leaf protoplast cells transformed with a promoterless construct.

Analysis of GUS Expression in Corn Leaf Protoplasts Driven by theSynthetic EXP, EXP-Zm.GSP990.

Corn leaf protoplast cells were transformed with expression vectors thatwere constructed comprising expression elements driving GUS expression.A test expression vector comprised a transgene cassette which comprisedthe synthetic EXP, EXP-Zm.GSP990 (SEQ ID NO:6), operably linked 5′ tothe intron I-Zm.DnaK:1 (SEQ ID NO:22), operably linked 5′ to a codingsequence encoding GUS (SEQ ID NO:20), which was operably linked 5′ tothe 3′ UTR, T-Os.LTP:1. The synthetic EXP-Zm.GSP990 (SEQ ID NO:6) iscomprised of a synthetic promoter, P-Zm.GSP990.nno:2 (SEQ ID NO:7),operably linked 5′ to a synthetic leader, L-Zm.GSP990.nno:1 (SEQ IDNO:8). Three control expression vectors were also transformed into cornleaf protoplasts and were constructed as described above. Table 3 showsthe mean percent expression relative to the third control expressionvector that comprises EXP-CaMV.35S and I-Zm.DnaK:1 driving GUS.

TABLE 3 Mean percent GUS expression of corn leaf protoplasts transformedwith test and control vectors. Plate ID Promoter Intron Mean SD Reps 71No Promoter I-Zm.DnaK: 1 0.3 0.125 6 108 No Promoter I-Zm.DnaK: 1 −0.61.211 8 71 EXP-CaMV.35S No Intron 45.1 7.791 6 108 EXP-CaMV.35S NoIntron 39.9 2.636 8 71 EXP-CaMV.35S I-Zm.DnaK: 1 100 13.591 6 108EXP-CaMV.35S I-Zm.DnaK: 1 100 7.425 8 71 EXP-Zm.GSP990 I-Zm.DnaK: 1 52.313.658 4 108 EXP-Zm.GSP990 I-Zm.DnaK: 1 45.2 5.468 8

As can be seen in Table 3, EXP-Zm.GSP990 (SEQ ID NO:6) was able to driveGUS transgene expression in corn leaf protoplasts when compared to cornleaf protoplast cells transformed with a promoterless construct.

Analysis of Enhancement of GUS Expression by the Synthetic Intron,I-Zm.GSI153.Nno:1

Corn leaf protoplast cells were transformed with expression vectors thatwere constructed comprising expression elements driving GUS expression.A test expression vector was used to assay the enhancement of GUSexpression from the synthetic intron, I-Zm.GSI153.nno:1 (SEQ ID NO:5),driven by EXP-CaMV.35. The transgene cassette comprised the EXP,EXP-CaMV.35 operably linked 5′ to the synthetic intron,I-Zm.GSI153.nno:1 (SEQ ID NO:5), operably linked 5′ to a coding sequenceencoding GUS (SEQ ID NO:24), which was operably linked 5′ to the 3′ UTR,T-Os.LTP:1. Two control expression vectors were also constructed andused to transform corn leaf protoplasts. The first control expressionvector comprised an intronless transgene cassette and was comprised ofthe EXP, EXP-CaMV.35S, operably linked 5′ to the GUS coding sequence,which was operably linked 5′ to the 3′ UTR, T-Os.LTP:1. The secondcontrol vector comprised a transgene cassette which comprised the EXP,EXP-CaMV.35S, operably linked 5′ to the intron I-Zm.DnaK:1, operablylinked 5′ to the GUS coding sequence, which was operably linked 5′ tothe 3′ UTR, T-Os.LTP:1. Table 4 shows the mean percent expressionrelative to the second control expression vector that comprises bothEXP-CaMV.35S and I-Zm.DnaK:1 driving GUS.

TABLE 4 Mean percent GUS expression of corn leaf protoplasts transformedwith test and control vectors. Plate ID Promoter Intron Mean SD Reps 10EXP-CaMV.35S No Intron 52.8 8.428 6 13 EXP-CaMV.35S No Intron 44.4 4.5868 10 EXP-CaMV.35S I-Zm.DnaK: 1 100 16.646 6 13 EXP-CaMV.35S I-Zm.DnaK: 1100 13.123 8 10 EXP-CaMV.35S I-Zm.GSI153.nno: 1 83 5.601 4 13EXP-CaMV.35S I-Zm.GSI153.nno: 1 83.6 7.596 8

As can be seen in Table 4, the synthetic intron, I-Zm.GSI153.nno:1 (SEQID NO:5), enhanced GUS transgene expression in corn leaf protoplastsdriven by EXP-CaMV.35S when compared to the intronless controlexpression vector.

Analysis of Enhancement of GUS Expression by the Synthetic Intron,I-Zm.GSI197.Nno:1

Corn leaf protoplast cells were transformed with expression vectors thatwere constructed comprising expression elements driving GUS expression.A test expression vector was used to assay the enhancement of GUSexpression from the synthetic intron, I-Zm.GSI197.nno:1 (SEQ ID NO:10),driven by EXP-CaMV.35. The transgene cassette comprised the EXP,EXP-CaMV.35 operably linked 5′ to the synthetic intron,I-Zm.GSI197.nno:1, operably linked 5′ to a coding sequence encoding GUS(SEQ ID NO:24), which was operably linked 5′ to the 3′ UTR, T-Os.LTP:1.Three control expression vectors were also constructed and used totransform corn leaf protoplasts. The first control expression vectorcomprised a promoterless transgene cassette and was comprised of theintron, I-Zm.DnaK:1, operably linked 5′ to the GUS coding sequence,which was operably linked 5′ to the 3′ UTR, T-Os.LTP:1. The secondcontrol vector comprised an intronless transgene cassette and wascomprised of the EXP, EXP-CaMV.35S, operably linked 5′ to the GUS codingsequence, which was operably linked 5′ to the 3′ UTR, T-Os.LTP:1. Thethird control vector comprised a transgene cassette which comprised theEXP, EXP-CaMV.35S, operably linked 5′ to the intron I-Zm.DnaK:1,operably linked 5′ to the GUS coding sequence, which was operably linked5′ to the 3′ UTR, T-Os.LTP:1. Table 5 shows the mean percent expressionrelative to the third control expression vector that comprises bothEXP-CaMV.35S and I-Zm.DnaK:1 driving GUS.

TABLE 5 Mean percent GUS expression of corn leaf protoplasts transformedwith test and control vectors. Plate ID Promoter Intron Mean SD Reps 125No Promoter I-Zm.DnaK: 1 1.1 0.317 6 146 No Promoter I-Zm.DnaK: 1 0.60.101 8 125 EXP-CaMV.35S No Intron 43.8 4.081 6 146 EXP-CaMV.35S NoIntron 41 3.666 8 125 EXP-CaMV.35S I-Zm.DnaK: 1 100 11.287 6 146EXP-CaMV.35S I-Zm.DnaK: 1 100 8.506 8 125 EXP-CaMV.35S I-Zm.GSI197.nno:1 150 12.451 4 146 EXP-CaMV.35S I-Zm.GSI197.nno: 1 109.8 8.001 8

As can be seen in Table 5, the synthetic intron, I-Zm.GSI197.nno:1 (SEQID NO:10), enhanced GUS transgene expression in corn leaf protoplastsdriven by EXP-CaMV.35S when compared to the intronless controlexpression vector. The enhancement of expression was greater than thatimparted by the intron, I-Zm.DnaK:1 when compared to the third controlexpression vector which comprised the EXP, EXP-CaMV.35S, operably linked5′ to the intron I-Zm.DnaK: 1.

Analysis of Enhancement of GUS Expression by the Synthetic 3′ UTRs,T-Zm.GST9.Nno:2 and T-Zm.GST18.nno:2.

Corn leaf protoplast cells were transformed with expression vectors thatwere constructed comprising expression elements driving GUS expression.Two test vectors comprised a transgene cassette used for the analysis ofGUS expression enhancement imparted by the 3′ UTRs, T-Zm.GST9.nno:2 (SEQID NO:13) and T-Zm.GST18.nno:2 (SEQ ID NO:14) and was comprised ofEXP-CaMV.35S operably linked 5′ to the intron I-Zm.DnaK:1, operablylinked 5′ to a coding sequence encoding GUS (SEQ ID NO:24), which wasoperably linked 5′ to the either the 3′ UTR, T-Zm.GST9.nno:2 (SEQ IDNO:13) or the 3′ UTR, T-Zm.GST18.nno:2 (SEQ ID NO:14). Three controlexpression vectors were also constructed, as described above, and usedto transform corn leaf protoplasts. Table 6 shows the mean percentexpression relative to the third control expression vector thatcomprises both EXP-CaMV.35S and I-Zm.DnaK:1 driving GUS.

TABLE 6 Mean percent GUS expression of corn leaf protoplasts transformedwith test and control vectors. Plate ID Promoter Intron 3′ UTR Mean SDReps 119 No Promoter I-Zm.DnaK:1 T-Os.LTP:1 3.5 2.163 8 184 No PromoterI-Zm.DnaK:1 T-Os.LTP:1 0.1 0.205 6 185 No Promoter I-Zm.DnaK:1T-Os.LTP:1 0 0.331 8 328 No Promoter I-Zm.DnaK:1 T-Os.LTP:1 10.2 3.017 6119 EXP-CaMV.35S No Intron T-Os.LTP:1 45.5 3.729 8 184 EXP-CaMV.35S NoIntron T-Os.LTP:1 33.7 2.719 6 185 EXP-CaMV.35S No Intron T-Os.LTP:133.6 2.576 8 328 EXP-CaMV.35S No Intron T-Os.LTP:1 48.8 3.69 6 119EXP-CaMV.35S I-Zm.DnaK:1 T-Os.LTP:1 100 7.305 8 184 EXP-CaMV.35SI-Zm.DnaK:1 T-Os.LTP:1 100 6.91 6 185 EXP-CaMV.35S I-Zm.DnaK:1T-Os.LTP:1 100 6.308 8 328 EXP-CaMV.35S I-Zm.DnaK:1 T-Os.LTP:1 100 5.9896 184 EXP-CaMV.35S I-Zm.DnaK:1 T-Zm.GST9.nno:2 202.5 17.582 4 119EXP-CaMV.35S I-Zm.DnaK:1 T-Zm.GST18.nno:2 483.2 40.613 8 185EXP-CaMV.35S I-Zm.DnaK:1 T-Zm.GST18.nno:2 254.5 18.347 8 328EXP-CaMV.35S I-Zm.DnaK:1 T-Zm.GST18.nno:2 307.4 17.772 4

As can be seen in Table 6, the 3′ UTRs, T-Zm.GST9.nno:2 (SEQ ID NO:13)and T-Zm.GST18.nno:2 (SEQ ID NO:14) enhanced GUS expression relative tothe controls in corn leaf protoplasts.

Example 3 Analysis of GUS Expression Driven by the Synthetic EXPs,EXP-Zm.GSP850.Nno+Zm.GSI153.Nno:2 and EXP-Zm.GSP850.Nno+Zm.GSI140.Nno:1in Stably Transformed LH244 Variety Corn Plants

Corn plants were transformed with a vector, specifically a plantexpression vector containing test regulatory elements driving expressionof the β-glucuronidase (GUS) transgene. The resulting plants wereanalyzed for GUS protein expression, to assess the effect of theselected regulatory element on expression.

Corn plants were transformed with plant GUS expression constructs. Theregulatory elements were cloned into a base plant expression vectorusing standard methods known in the art. The resulting plant expressionvectors contained a left border region from Agrobacterium tumefaciens(B-AGRtu.left border), a first transgene selection cassette used forselection of transformed plant cells that confers resistance to theherbicide glyphosate; a second transgene cassette to assess the activityof the synthetic regulatory elements, which comprised either thesynthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) orEXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11) operably linked 5′ to asynthetic coding sequence designed for expression in a plant cellencoding β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20)containing a processable intron derived from the potato light-inducibletissue-specific ST-LS1 gene (Genbank Accession: X04753), operably linked5′ to a 3′ termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO:19); and aright border region from Agrobacterium tumefaciens (B-AGRtu.rightborder). The synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ IDNO:4) is comprised of a synthetic promoter, P-Zm.GSP850.nno:4 (SEQ IDNO:2), operably linked 5′ to a synthetic leader, L-Zm.GSP850.nno:3 (SEQID NO:3), which is operably linked 5′ to a synthetic intron,I-Zm.GSI153.nno:1 (SEQ ID NO:5). The synthetic EXP,EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11) is comprised of asynthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO:2), operably linked 5′to a synthetic leader, L-Zm.GSP850.nno:3 (SEQ ID NO:3), which isoperably linked 5′ to a synthetic intron, I-Zm.GSI140.nno:1 (SEQ IDNO:12).

Corn variety LH244 plant cells were transformed using the binarytransformation vector construct described above byAgrobacterium-mediated transformation, as is well known in the art. Theresulting transformed plant cells were induced to form whole cornplants.

Qualitative and quantitative GUS analysis was used to evaluateexpression element activity in selected plant organs and tissues intransformed plants. For qualitative analysis of GUS expression byhistochemical staining, whole-mount or sectioned tissues were incubatedwith GUS staining solution containing 1 mg/mL of X-Gluc(5-bromo-4-chloro-3-indolyl-b-glucuronide) for 5 h at 37° C. andde-stained with 35% EtOH and 50% acetic acid. Expression of GUS wasqualitatively determined by visual inspection of selected plant organsor tissues for blue coloration under a dissecting or compoundmicroscope.

For quantitative analysis of GUS expression by enzymatic assays, totalprotein was extracted from selected tissues of transformed corn plants.One to two micrograms of total protein was incubated with thefluorogenic substrate, 4-methyleumbelliferyl-β-D-glucuronide (MUG) at 1mM concentration in a total reaction volume of 50 microliters. After 1 hincubation at 37° C., the reaction was stopped by adding 350 microlitersof 200 mM sodium bicarbonate solution. The reaction product,4-methlyumbelliferone (4-MU), is maximally fluorescent at high pH, wherethe hydroxyl group is ionized. Addition of the basic sodium carbonatesolution simultaneously stops the assay and adjusts the pH forquantifying the fluorescent product 4-MU. The amount of 4-MU formed wasestimated by measuring its fluorescence using a FLUOstar OmegaMicroplate Reader (BMG LABTECH) (excitation at 355 nm, emission at 460nm). GUS activity values are provided in nmoles of 4-MU/hour/mg totalprotein.

The following tissues were sampled for GUS expression in the R₀generation: V4 stage Leaf and Root; V7 stage Leaf and Root; VT stageLeaf, Root, and Flower/Anther; R1 stage Cob/Silk; and R3 stage SeedEmbryo and Seed Endosperm 21 days after pollination (DAP). Table 7 showsthe mean quantitative GUS expression values for each of the syntheticEXPs.

TABLE 7 Mean quantitative GUS expression in stably transformed LH244variety corn plants driven by the synthetic EXPs, EXP-Zm.GSP850.nno +Zm.GSI153.nno: 2 and EXP-Zm.GSP850.nno + Zm.GSI140.nno: 1. EXP- EXP-Zm.GSP850.nno + Zm.GSP850.nno + Zm.GSI153.nno: 2 Zm.GSI140.nno: 1 StageOrgan (SEQ ID NO: 4) (SEQ ID NO: 11) V4 Leaf 694 642 Root 870 116 V7Leaf 1423 1265 Root 476 521 VT Leaf 2646 344 Root 424 70 Flower/Anther5465 1267 R1 Cob/Silk 3618 1116 R3 Seed Embryo 260 224 21 DAP SeedEndosperm 2648 1140 21 DAP

As can be seen in Table 7, the synthetic GSP850 promoter and leader(P-Zm.GSP850.nno:4 (SEQ ID NO:2) and L-Zm.GSP850.nno:3 (SEQ ID NO:3))drove constitutive expression of GUS in stably transformed LH244 varietycorn plants. Molecular analysis of the transcript start sitedemonstrated a consistent TSS for the GSP850 promoter and leader. Thesynthetic introns, I-Zm.GSI153.nno:1 (SEQ ID NO:5) and I-Zm.GSI140.nno:1(SEQ ID NO:12) affected expression differently in the different tissuessampled. Molecular analysis of the intron splice sites demonstratedconsistent processing of the synthetic introns. Overall enhancement ofGUS expression was higher in most tissue samples from plants comprisingI-Zm.GSI153.nno:1 (SEQ ID NO:5), with the exception of V4 stage leaf, V7stage root, and R3 seed embryo where GUS expression levels wererelatively similar. Enhancement of expression imparted byI-Zm.GSI153.nno:1 (SEQ ID NO:5) relative to I-Zm.GSI140.nno:1 (SEQ IDNO:12) was approximately 7.5-fold higher in V4 root, 7.7 fold higher inVT leaf, 6.0 fold higher in VT root, 4.3 fold higher in VTflower/anther, 3.2 fold higher in R1 cob/silk, and 2.3 fold higher in R3seed endosperm.

Example 4 Analysis of GUS Expression Driven by the Synthetic EXPsEXP-Zm.GSP850.Nno+Zm.DnaK:1, EXP-Zm.GSP850.Nno+Zm.GSI153.Nno:2, andEXP-Zm.GSP850.Nno+Zm.GSI140.Nno:1 in Stably Transformed 01DKD2 VarietyCorn Plants

Corn plants were transformed with a vector, specifically a plantexpression vector containing test regulatory elements driving expressionof the β-glucuronidase (GUS) transgene. The resulting plants wereanalyzed for GUS protein expression, to assess the effect of theselected regulatory element on expression.

Corn plants were transformed with plant GUS expression constructs. Theregulatory elements were cloned into a base plant expression vectorusing standard methods known in the art. The resulting plant expressionvectors contained a left border region from Agrobacterium tumefaciens(B-AGRtu.left border), a first transgene selection cassette used forselection of transformed plant cells that confers resistance to theherbicide glyphosate; a second transgene cassette to assess the activityof the regulatory elements, which comprised either the synthetic EXP,EXP-Zm.GSP850.nno+Zm.DnaK:1 (SEQ ID NO:15),EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) orEXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11) operably linked 5′ to asynthetic coding sequence designed for expression in a plant cellencoding β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20)containing a processable intron derived from the potato light-inducibletissue-specific ST-LS1 gene (Genbank Accession: X04753), operably linked5′ to a 3′ termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO:19); and aright border region from Agrobacterium tumefaciens (B-AGRtu.rightborder). The synthetic EXP, EXP-Zm.GSP850.nno+Zm.DnaK:1 (SEQ ID NO:15)is comprised of a synthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO:2),operably linked 5′ to a synthetic leader, L-Zm.GSP850.nno:3 (SEQ IDNO:3), which is operably linked 5′ to an intron, I-Zm.DnaK:1 (SEQ ID NO:22). The synthetic EXPs, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4)and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1(SEQ ID NO:11) are described inExample 3.

Corn variety 01DKD2 plant cells were transformed using the binarytransformation vector construct described above byAgrobacterium-mediated transformation, as is well known in the art. Theresulting transformed plant cells were induced to form whole cornplants. Qualitative and quantitative GUS expression was assayed asdescribed in Example 3. Table 8 shows the mean quantitative GUSexpression values for each of the synthetic EXPs.

TABLE 8 Mean quantitative GUS expression in stably transformed 01DKD2variety corn plants driven by the synthetic EXPs EXP-Zm.GSP850.nno +Zm.DnaK: 1, EXP-Zm.GSP850.nno + Zm.GSI153.nno: 2, andEXP-Zm.GSP850.nno + Zm.GSI140.nno: 1. EXP- EXP- EXP- Zm.GSP850.nno +Zm.DnaK: 1 Zm.GSP850.nno + Zm.GSI153.nno: 2 Zm.GSP850.nno +Zm.GSI140.nno: 1 Stage Organ (SEQ ID NO: 15) (SEQ ID NO: 4) (SEQ ID NO:11) V4 Leaf 208 374 519 Root 36 606 341 V7 Leaf 314 539 619 Root 22 1158303 VT Leaf 26 610 630 Root 32 770 797 Flower/Anther 104 797 1121 R1Cob/Silk 160 1139 1312 R3 Seed Embryo 31 188 723 21 DAP Seed Endosperm98 1490 1635 21 DAP

As can be seen in Table 8, the synthetic GSP850 promoter and leader(P-Zm.GSP850.nno:4 (SEQ ID NO:2) and L-Zm.GSP850.nno:3 (SEQ ID NO:3))drove constitutive expression of GUS in stably transformed LH244 varietycorn plants. The synthetic introns, I-Zm.GSI153.nno:1 (SEQ ID NO:5) andI-Zm.GSI140.nno:1 (SEQ ID NO:12) enhanced expression relative to theintron, I-Zm.DnaK:1 (SEQ ID NO:22) in all tissues assayed.

Example 5 Analysis of GUS Expression Driven by the Synthetic EXPsEXP-Zm.GSP990.Nno+Zm.DnaK:1 and EXP-Zm.GSP990.Nno+Zm.GSI197.Nno:2 inStably Transformed 01DKD2 Variety Corn Plants

Corn plants were transformed with a vector, specifically a plantexpression vector containing test regulatory elements driving expressionof the β-glucuronidase (GUS) transgene. The resulting plants wereanalyzed for GUS protein expression, to assess the effect of theselected regulatory element on expression.

Corn plants were transformed with plant GUS expression constructs. Theregulatory elements were cloned into a base plant expression vectorusing standard methods known in the art. The resulting plant expressionvectors contained a left border region from Agrobacterium tumefaciens(B-AGRtu.left border), a first transgene selection cassette used forselection of transformed plant cells that confers resistance to theherbicide glyphosate; a second transgene cassette to assess the activityof the regulatory elements, which comprised either the synthetic EXPEXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO:16) orEXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO:9) operably linked 5′ to asynthetic coding sequence designed for expression in a plant cellencoding β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20)containing a processable intron derived from the potato light-inducibletissue-specific ST-LS1 gene (Genbank Accession: X04753), operably linked5′ to a 3′ termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO:19); and aright border region from Agrobacterium tumefaciens (B-AGRtu.rightborder). The synthetic EXP EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ IDNO:9) is comprised of a synthetic promoter, P-Zm.GSP990.nno:2 (SEQ IDNO:7), operably linked 5′ to a synthetic leader, L-Zm.GSP990.nno:1 (SEQID NO:8), which is operably linked 5′ to a synthetic intron,I-Zm.GSI197.nno:1 (SEQ ID NO:10). The synthetic EXPEXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO:16) is comprised of a syntheticpromoter, P-Zm.GSP990.nno:2 (SEQ ID NO:7), operably linked 5′ to asynthetic leader, L-Zm.GSP990.nno:1 (SEQ ID NO:8), which is operablylinked 5′ to an intron, I-Zm.DnaK:1 (SEQ ID NO:22).

Corn variety 01DKD2 plant cells were transformed using the binarytransformation vector construct described above byAgrobacterium-mediated transformation, as is well known in the art. Theresulting transformed plant cells were induced to form whole cornplants. Qualitative and quantitative GUS expression was assayed aspreviously described in Example 3. Table 9 shows the mean quantitativeGUS expression values for each of the synthetic EXPs, wherein “ND”indicates not determined.

TABLE 9 Mean quantitative GUS expression in stably transformed 01DKD2variety corn plants driven by the synthetic EXPs EXP-Zm.GSP990.nno +Zm.DnaK: 1 and EXP-Zm.GSP990.nno + Zm.GSI197.nno: 2. EXP- EXP-Zm.GSP990.nno + Zm.GSP990.nno + Zm.DnaK: 1 Zm.GSI197.nno: 2 Stage Organ(SEQ ID NO: 16) (SEQ ID NO: 9) V4 Leaf 99 75 Root 32 33 V7 Leaf 226 138Root 29 40 VT Leaf 140 61 Root 55 96 Flower/Anther 87 231 R1 Cob/Silk 3935 R3 Seed Embryo ND 21 21 DAP Seed Endosperm ND 22 21 DAP

As can be seen, the synthetic GSP990 promoter and leader(P-Zm.GSP990.nno:2 (SEQ ID NO:7) and L-Zm.GSP990.nno:1 (SEQ ID NO:8))drove expression of GUS. Molecular analysis of the transcript start sitedemonstrated a consistent TSS for the GSP990 promoter and leader. Thesynthetic intron, I-Zm.GSI197.nno:1 (SEQ ID NO:10) attenuated expressionin some tissues, while enhancing expression in other tissues, relativeto the intron, I-Zm.DnaK:1 (SEQ ID NO:22). For example, expression ofGUS was attenuated in leaf at V4, V7, and VT stage. GUS expression wasslightly enhanced in the V7 and VT root, relative to I-Zm.DnaK:1.Flower/anther expression was enhanced approximately 2.7 fold byI-Zm.GSI197.nno:1 (SEQ ID NO:10), relative to I-Zm.DnaK: 1. Thedifferences in expression imparted by I-Zm.GS1197.nno:1 (SEQ ID NO:10),relative to I-Zm.DnaK:1, can be very useful where lower leaf and higherflower/anther expression is desired. Molecular analysis of the intronsplice sites demonstrated consistent processing of the synthetic intron,I-Zm.GSI197.nno:1 (SEQ ID NO:10).

Example 6 Analysis of the Effect on GUS Expression by the Synthetic 3′UTRs T-Zm.GST9.Nno:2, T-Zm.GST18.Nno:2, and T-Zm.GST43.Nno:1, and theNative T-Sb.Ntlp4-1:1:2 in Stably Transformed 01DKD2 Variety Corn Plants

Corn plants were transformed with a vector, specifically a plantexpression vector containing test regulatory elements driving expressionof the β-glucuronidase (GUS) transgene. The resulting plants wereanalyzed for GUS protein expression, to assess the effect of theselected regulatory element on expression.

Corn plants were transformed with plant GUS expression constructs. Theregulatory elements were cloned into a base plant expression vectorusing standard methods known in the art. The resulting plant expressionvectors contained a left border region from Agrobacterium tumefaciens(B-AGRtu.left border), a first transgene selection cassette used forselection of transformed plant cells that confers resistance to theherbicide glyphosate; a second transgene cassette to assess the activityof the 3′ UTR regulatory elements, which comprised the EXP EXP-CaMV.35S(SEQ ID NO:21), operably linked 5′ to the intron, I-Zm.DnaK:1 (SEQ IDNO:22), operably linked 5′ to a synthetic coding sequence designed forexpression in a plant cell encoding β-glucuronidase (GUS,GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20) containing a processable intronderived from the potato light-inducible tissue-specific ST-LS1 gene(Genbank Accession: X04753), operably linked 5′ to a 3′ terminationregion; and a right border region from Agrobacterium tumefaciens(B-AGRtu.right border). Three test expression vectors comprised the 3′UTRs T-Zm.GST9.nno:2 (SEQ ID NO:13), T-Zm.GST18.nno:2 (SEQ ID NO:14), orT-Zm.GST43.nno:1 (SEQ ID NO:26) operably linked to the GUS codingsequence. An additional test expression vector comprised the native 3′UTR T-Sb.Nltp4-1:1:2 (SEQ ID NO:19) operably linked to the GUS codingsequence, and was used to compare expression between native andsynthetic 3′ UTRs.

Corn variety 01DKD2 plant cells were transformed using the binarytransformation vector constructs described above byAgrobacterium-mediated transformation, as is well known in the art. Theresulting transformed plant cells were induced to form whole cornplants.

Qualitative and quantitative GUS analysis was used to evaluateexpression element activity in V4 leaf and root tissues of thetransformed plants and was performed as described above in Example 3.The effect of the synthetic 3′ UTRs was assessed through comparison tothe effect of expression from the 3′ UTR T-Sb.Nltp4-1:1:2 (SEQ IDNO:19). The resulting transcripts were analyzed to determine if propertermination had occurred and that there was no read through of thetranscript. One method to assess if there was read through was throughthe use of amplification of transcript cDNA using an amplificationprimer corresponding to a portion of the T-DNA border sequence that is3′ to the 3′ UTR. The mean GUS expression of plants transformed with thefour constructs comprising T-Zm.GST9.nno:2 (SEQ ID NO:13),T-Zm.GST18.nno:2 (SEQ ID NO:14), T-Zm.GST43.nno:1 (SEQ ID NO:26), andT-Sb.Nltp4-1:1:2 (SEQ ID NO:19) is provided in Table 10.

TABLE 10 Mean quantitative GUS expression in stably transformed 01DKD2variety corn plants from constructs comprising three different 3′ UTRs.T-Sb.Nltp4-1: 1: 2 T-Zm.GST9.nno: 2 T-Zm.GST18.nno: 2 T-Zm.GST43.nno: 1Stage Organ (SEQ ID NO: 19) (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO:26) V4 Leaf 350 684 423 113 Root 778 924 810 883

As can be seen in Table 10, both T-Zm.GST9.nno:2 (SEQ ID NO:13) andT-Zm.GST18.nno:2 (SEQ ID NO:14) enhanced GUS expression driven byEXP-CaMV.35S operably linked I-Zm.DnaK:1, relative to the 3′ UTR,T-Sb.Nltp4-1:1:2. GUS expression in plants comprising T-Zm.GST9.nno:2(SEQ ID NO:13) was higher than plants comprising T-Zm.GST18.nno:2.T-Zm.GST43.nno:1 (SEQ ID NO:26) enhanced GUS expression in the V4 rootrelative to the T-Sb.Nltp4-1:1:2, but attenuated expression in the V4leaf. Analysis of the GUS transcripts from all four constructsdemonstrated proper termination of the transcript and no evidence ofread through in the resulting GUS transcripts. The 3′ UTRsT-Zm.GST9.nno:2 (SEQ ID NO:13), T-Zm.GST18.nno:2 (SEQ ID NO:14), andT-Zm.GST43.nno:1 (SEQ ID NO:26) operate in a similar manner as native 3′UTRs and demonstrated modulation of GUS expression, relative to thenative 3′ UTR T-Sb.Nltp4-1:1:2. All four synthetic 3′ UTRs and theadditional native 3′ UTR T-Sb.Nltp4-1:1:2 provide a range of expressionvalues that are useful in fine-tuning expression in stably transformedcorn plants.

Example 7 Enhancer Elements Derived from the Regulatory Elements

Enhancers are derived from the promoter elements presented as SEQ IDNOs:2 and 7. The enhancer elements may be comprised of one or more cisregulatory elements that when operably linked 5′ or 3′ to a promoterelement, or operably linked 5′ or 3′ to additional enhancer elementsthat are operably linked to a promoter, can enhance or modulateexpression levels of a transcribable DNA molecule, or provide expressionof a transcribable DNA molecule in a specific cell type or plant organor at a particular time point in development or circadian rhythm.Enhancers are made by removing the TATA box or functionally similarelements and any downstream sequence from the promoters that allowtranscription to be initiated from the promoters presented as SEQ IDNOs:2 and 7 or fragments thereof.

The TATA box in plant promoters is not as highly conserved as in someother eukaryotic organisms. Therefore, in order to define a fragment asan enhancer, one first must identify the transcriptional start site(TSS) of the gene, wherein the 5′ UTR is first transcribed. An enhancerderived from the synthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO:2)could comprise nucleotides 1 through 418 of SEQ ID NO:2, resulting inthe synthetic enhancer, E-Zm.GSP850 (SEQ ID NO:17). An enhancer derivedfrom the synthetic promoter, P-Zm.GSP990.nno:2 (SEQ ID NO:7) couldcomprise nucleotides 1 through 416 of SEQ ID NO:7, resulting in thesynthetic enhancer, E-Zm.GSP990 (SEQ ID NO:18). Enhancers derived fromthe promoters may comprise fragments of SEQ ID NOs:17 and 18, orduplications of SEQ ID NOs:17 and 18 or their respective fragments. Theeffectiveness of the synthetic enhancers derived from the syntheticpromoters is empirically determined by building a chimerictranscriptional regulatory element comprising fragments derived fromeither the synthetic promoters P-Zm.GSP850.nno:4 (SEQ ID NO:2) orP-Zm.GSP990.nno:2 (SEQ ID NO:7), which is operably linked to a promoterand leader and used to drive expression of a transcribable DNA moleculesuch as GUS in stable or transient plant assay.

Further refinement of the enhancer element may be required and isvalidated empirically. In addition, position of the enhancer elementrelative to other elements within a chimeric transcriptional regulatoryelement is also empirically determined, since the order of each elementwithin the chimeric transcriptional regulatory element may impartdifferent effects, depending upon the relative positions of eachelement. Some promoter elements will have multiple TATA box or TATAbox-like elements and potentially multiple transcription start sites.Under those circumstances, it may be necessary to first identify wherethe first TSS is located and then begin designing enhancers using thefirst TSS to prevent the potential initiation of transcription fromoccurring within a putative enhancer element.

Enhancer elements, derived from the promoter elements presented as SEQID NOs:2 and 7, are cloned using methods known in the art to be operablylinked 5′ or within a promoter element, or operably linked 5′ or 3′ toadditional enhancer elements that are operably linked to a promoter.Alternatively, enhancer elements can be cloned, using methods known inthe art, to provide a larger enhancer element that is comprised of twoor more copies of the enhancer and cloned using methods known in the artto be operably linked 5′ or 3′ to a promoter element, or operably linked5′ or 3′ to additional enhancer elements that are operably linked to apromoter producing a chimeric transcriptional regulatory element.Enhancer elements derived from promoters derived from genes frommultiple genus organisms can be operably linked to the enhancers derivedfrom the synthetic promoters.

A GUS expression plant transformation vector may be constructed usingmethods known in the art similar to the construct described in Example 3in which the resulting plant expression vectors contain a left borderregion from Agrobacterium tumefaciens (B-AGRtu.left border), a firsttransgene selection cassette used for selection of transformed plantcells that confers resistance to the herbicide glyphosate; and a secondtransgene cassette to test the enhancer element comprised of theenhancer element operably linked 5′ or 3′ to a promoter element oroperably linked 5′ or 3′ to additional enhancer elements that are inturn operably linked to a promoter which is operably linked 5′ to aleader element, operably linked 5′ to an intron element, operably linkedto a coding sequence for β-glucuronidase (GOI-Ec.uidA+St.LS1.nno:1, SEQID NO:20) containing a processable intron derived from the potatolight-inducible tissue-specific ST-LS1 gene (Genbank Accession: X04753),operably linked to a 3′ termination region from the Oryza sativa LipidTransfer Protein-like gene (T-Os.LTP:1, SEQ ID NO:23); and a rightborder region from A. tumefaciens (B-AGRtu.right border). The resultingplasmids are used to transform corn plants or other monocot genus plantsby the methods described above. Alternatively, protoplast cells derivedfrom corn or other monocot genus plants are transformed using methodsknown in the art to perform transient assays

GUS expression driven by a regulatory element comprising one or moreenhancers is evaluated in stable or transient plant assays to determinethe effects of the enhancer element on expression of a transcribable DNAmolecule. Modifications to one or more enhancer elements or duplicationof one or more enhancer elements may be performed based upon empiricalexperimentation and the resulting gene expression regulation that isobserved using each regulatory element composition. Altering therelative positions of one or more enhancers in the resulting regulatoryor chimeric regulatory elements may affect the transcriptional activityor specificity of the regulatory or chimeric regulatory element and isdetermined empirically to identify the best enhancers for the desiredtransgene expression profile within the corn plant or other genus plant.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the claims. All publications and published patentdocuments cited herein are hereby incorporated by reference to the sameextent as if each individual publication or patent application isspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A recombinant DNA molecule comprising a DNAsequence selected from the group consisting of: a) a sequence comprisingSEQ ID NO:26; and b) a fragment comprising at least 200 contiguousnucleotides of SEQ ID NO:26, wherein the fragment has gene-regulatoryactivity; wherein said DNA sequence is operably linked to a heterologoustranscribable DNA molecule.
 2. The recombinant DNA molecule of claim 1,wherein said sequence comprises at least 225 contiguous nucleotides ofSEQ ID NO:26.
 3. The recombinant DNA molecule of claim 1, wherein saidsequence comprises at least 250 contiguous nucleotides of SEQ ID NO:26.4. The recombinant DNA molecule of claim 1, wherein the DNA sequencecomprises gene regulatory activity.
 5. The recombinant DNA molecule ofclaim 1, wherein the heterologous transcribable DNA molecule comprises agene of agronomic interest.
 6. The recombinant DNA molecule of claim 5,wherein the gene of agronomic interest confers herbicide tolerance inplants.
 7. The recombinant DNA molecule of claim 5, wherein the gene ofagronomic interest confers pest resistance in plants.
 8. The recombinantDNA molecule of claim 1, wherein the heterologous transcribable DNAmolecule encodes a dsRNA, an miRNA, or a siRNA.
 9. A transgenic plantcell comprising a recombinant DNA molecule comprising a DNA sequenceselected from the group consisting of: a) a sequence comprising SEQ IDNO:26; and b) a fragment comprising at least 200 contiguous nucleotidesof SEQ ID NO:26, wherein the fragment has gene-regulatory activity;wherein said DNA sequence is operably linked to a heterologoustranscribable DNA molecule.
 10. The transgenic plant cell of claim 9,wherein said transgenic plant cell is a monocotyledonous plant cell. 11.The transgenic plant cell of claim 9, wherein said transgenic plant cellis a dicotyledonous plant cell.
 12. A transgenic plant, or part thereof,comprising the recombinant DNA molecule of claim
 1. 13. A progeny plantof the transgenic plant of claim 12, or a part thereof, wherein theprogeny plant or part thereof comprises said recombinant DNA molecule.14. A transgenic seed, wherein the seed comprises the recombinant DNAmolecule of claim
 1. 15. A method of producing a commodity productcomprising obtaining a transgenic plant or part thereof according toclaim 12 and producing the commodity product therefrom.
 16. The methodof claim 15, wherein the commodity product is seeds, processed seeds,protein concentrate, protein isolate, starch, grains, plant parts, seedoil, biomass, flour or meal.
 17. A method of expressing a transcribableDNA molecule comprising obtaining a transgenic plant according to claim12 and cultivating the plant, wherein the transcribable DNA isexpressed.
 18. The recombinant DNA molecule of claim 1, wherein said DNAsequence comprises SEQ ID NO:26.
 19. The transgenic plant cell of claim9, wherein said DNA sequence comprises SEQ ID NO:26.